Adenovirus-mediated intratumoral delivery of an angiogenesis antagonist for the treatment of tumors

ABSTRACT

The present invention relates to gene therapy for the treatment of tumors. The invention more particularly relates to introduction of a gene encoding an anti-angiogenic factor into cells of a tumor, for example with a defective adenovirus vector, to inhibit growth or metastasis, or both, of the tumor. In a specific embodiment, delivery of a defective adenovirus that expresses the amino terminal fragment of urokinase (ATF) inhibited growth and metastasis of tumors. These effects were correlated with a remarkable inhibition of neovascularization within, and at the immediate vicinity of, the injection site. Delivery of a defective adenovirus vector that expresses kringles 1 to 3 of angiostatin inhibited tumor growth and tumorigenicity, and induced apoptosis of tumor cells. The invention further provides viral vectors for use in the methods of the invention.

FIELD OF THE INVENTION

[0001] The present invention relates to gene therapy for the treatmentof tumors. The invention more particularly relates to introduction of agene encoding an anti-angiogenic factor into cells of a tumor, forexample with an adenovirus vector, to inhibit growth or metastasis, orboth, of the tumor.

BACKGROUND OF THE INVENTION

[0002] Cell migration is a coordinated process that bridges cellularactivation and adhesion whereas the equilibrium between pericellularproteolysis and its inhibition (e.g., triggered by plasminogen activatorinhibitors and tissue inhibitors of metalloproteinases) is disrupted(1-3). Urokinase plasminogen activator (uPA) is a pivotal player in thisprocess because it initiates a proteolytic cascade at the surface ofmigrating cells by binding to its cell surface receptor (uPAR) (4, 5).Binding of uPA to its receptor greatly potentiates plasminogen/plasminconversion at the cell surface (6). Plasmin is a broadly specific serineprotease which can directly degrade components of the extracellularmatrix such as fibronectin, vitronectin or laminin. Plasmin alsoindirectly promotes a localized degradation of the stroma by convertinginactive zymogens into active metalloproteinases (7). The selectivedistribution of uPAR at the leading edge of migrating cells(invadopodes) apparently concentrates uPA secreted by themselves or byneighboring stroma cells (8). uPAR is also directly involved in cellularadhesion to the extracellular matrix as illustrated by its uPA-dependentbinding to vitronectin (9), and because uPAR modulates the bindingproperties of several integrin molecules (10). Finally, uPA and plasminare somehow involved in cell morphogenesis by activating or inducing therelease of morphogenic factors such as vascular endothelial growthfactor (VEGF), hepatocyte growth factor (HGF), fibroblast growth factors(FGFs) and transforming growth factor B (TGFβ) (11, 12).

[0003] Taken together, these observations indicate that the uPA/uPARsystem controls cell migration by coordinating cellular activation,adhesion and motility. This statement is supported by clinicalobservations that correlate the presence of enhanced uPA activity at theinvasive edge of the tumors (13, 14). That melanoma induced by DMBA andcroton oil do not progress to a malignant stage in uPA-deficient micealso support a role of uPA in tumor establishment and progression (15).

[0004] uPA binds to uPAR by its light chain fragment, also known asamino-terminal fragment (ATF, amino acid 1-135). This interaction isspecies restricted (16) and involves the EGF-like domain of ATF(residues 146), in which amino acid 19-32, which are not conservedbetween mice and human, are critical (17, 18). ATF-mediated disruptionof the uPA/uPAR complex inhibits tumor cell migration and invasion invitro (19). Intraperitoneal bolus injection of a chimeric humanATF-based antagonist has also been used to inhibit lung metastases ofhuman tumor cells implanted within athymic mice, without significantlyaffecting primary tumor growth (20). A further study reported thatintraperitoneal injection of synthetic peptides derived from murine ATFwas effective in inhibiting both primary tumor growth and lungmetastases (21). These results are consistent with a role of theuPA/uPAR complex in controlling the motility of both tumor andendothelial cells (22). That a chimeric murine ATF-based antagonistcould inhibit vessel growth in an artificial bFGF-enriched extracellularmatrix (23) further supports uPA/uPAR involvement in controllingangiogenesis in vivo.

[0005] The formation of blood vessels, or angiogenesis, results from thecapillary growth of pre-existing vessels. Angiogenesis is essential fora number of physiological processes such as embryonic development, woundhealing and tissue or organ regeneration. Abnormal growth of new bloodvessels occurs in pathological conditions such as diabetic retinopathyand tumor growth, as well as tumor dissemination to distant sites[38,24]. Both experimental and clinical studies have showed that primarytumors as well as metastasis remain dormant due to a balanced rate ofproliferation and apoptosis unless the angiogenesis process is switchedon [39].

[0006] The growth of endothelial cells is tightly regulated by bothpositive and negative factors. The onset of tumor angiogenesis could betriggered either by an upregulation of tumor-released angiogenic factorssuch as vascular endothelial growth factor (VEGF) and acid or/and basicfibroblast growth factor (bFGFs), or by a downregulation of angiostaticfactors such as thrombospondin and angiostatin [39]. Both thereconstitution of angiostatic factors and the removal of angiogenicstimulating factors thus constitute plausible clinical strategies tosuppress tumor angiogenesis [40, 41]. Angiostatic-based therapies shouldalso apply to all solid tumors because endothelial cells do not varyfrom one tumor type to the other, further emphasizing the clinicalrelevance of such an anti-cancer approach. Thus, the therapy targetingangiogenesis appears to be highly relevant to clinical practice.

[0007] Many physiological angiostatic factors are derived uponproteolytic cleavage of circulating proteins. This is the case forangiostatin [32], endostatin [42], the 16 kDa fragment of prolactin[43], or platelet factor-4 [44]. Angiostatin was initially isolated frommice bearing a Lewis lung carcinoma (LLC), and was identified as a 38kDa internal fragment of plasminogen (Plg) (aa 98-440) that encompassesthe first four kringles of the molecule [32; WO95/29242; U.S. Pat. No.5,639,725]. Angiostatin has been shown to be generated followinghydrolysis of Plg by a metalloelastase from GM-CSF-stimulatedtumor-infiltrating macrophages [45]. Intraperitoneal bolus injections ofpurified angiostatin in six different tumor models have proved to bevery effective in suppressing primary tumor growth, with no apparenttoxicity [46]. Angiostatin-mediated suppression of tumor angiogenesisapparently drove the tumor cells into a higher apoptotic rate thatcounterbalanced their proliferation rate. In this study, tumor growthusually resumed following removal of the angiostatin molecule,emphasizing the importance of achieving long-term delivery for optimalclinical benefits [46]. In vitro studies with recombinant proteinsindicated that the angiostatic activity of angiostatin was mostlymediated by kringles 1-3, thus leaving a minor activity for kringle 4[47]. As for most angiostatic factors, little is known about themolecular pathway by which angiostatin exerts its effect.

[0008] As angiostatic therapy will require a prolonged maintenance oftherapeutic levels in vivo, the continuous delivery of a recombinantprotein will be expensive and cumbersome. Direct in vivo delivery of thecorresponding genes with viral vectors constitutes an attractivesolution to this problem. Because most cancer gene therapies currentlyrely on destructive strategies that target the tumor cells [48],viral-mediated gene delivery of an angiostatic factor represents aconceptually different, and possibly synergistic, approach to fightcancer.

[0009] Despite these results, there remains a need to develop effectivetreatments for tumors, particularly chemotherapy-resistant tumors.

[0010] The present invention addresses this need by establishing aneffective mode for treating a tumor.

[0011] Various references are cited in this specification by number,which are fully set forth after the Examples. None of the referencescited herein should be construed as describing or suggesting theinvention disclosed herein.

SUMMARY OF THE INVENTION

[0012] The present invention advantageously provides a highly effectivegene therapy for tumors. Indeed, in a specific embodiment of theinvention murine urokinase ATF expressed by human tumor cells in anathymic murine model unexpectedly effectively inhibits tumorigenicity.In another embodiment, angiostatin expressed in tumor cells in a murinemodel inhibited tumor growth and tumorigenesis, and induced tumor cellapoptosis, in addition to blocking angiogenesis.

[0013] In a broad aspect, the present invention provides a method forinhibiting growth or metastasis, or both, of a tumor comprisingintroducing a vector comprising a gene encoding an anti-angiogenicfactor operably associated with an expression control sequence thatprovides for expression of the anti-angiogenic factor into a cell orcells of the tumor. Preferably, the vector is a virus vector; morepreferably the virus vector is an adenovirus vector. In a specificembodiment exemplified infra, the adenovirus vector is a defectiveadenovirus vector.

[0014] The methods of the invention are useful in the treatment of manytumors, as set forth in detail herein. For example, in specificembodiments, the tumor is a lung carcinoma or a breast carcinoma.

[0015] In addition, the invention demonstrates for the first time theadvantages of expression of an anti-angiogenic factor by the transducedtumor cells. Accordingly, a gene encoding any anti-angiogenic factor,such as a soluble receptor for an angiogenic protein, or an angiogenesisantagonist, can be delivered in the practice of the invention. In aspecific embodiment, the anti-angiogenic factor comprises a sequence ofan amino terminal fragment of urokinase having an EGF-like domain, withthe proviso that the factor is not urokinase. For example, theanti-angiogenic factor may be a chimeric protein comprisingATF-immunoglobulin or ATF-human serum albumin. In a preferredembodiment, exemplified infra, the anti-angiogenic factor is an aminoterminal fragment of urokinase having an amino acid sequence ofurokinase from about amino acid residue 1 to about residue 135. In aspecific aspect, the urokinase is murine urokinase. In a more preferredaspect, the urokinase is human urokinase.

[0016] In an alternative embodiment, the anti-angiogenic factor isangiostatin, in particular, kringles 1 to 3 of angiostatin. In aparticularly preferred embodiment, the anti-angiogenic factor is theamino-terminal fragment of plasminogen (Plg) having an amino acidsequence of plasminogen from about amino acid residue 1 to about residue333. In another preferred embodiment, the anti-angiogenic factor is theamino-terminal fragment (angiostatin) from human plasminogen.

[0017] In a related embodiment, the invention is directed to use of avector comprising a gene encoding an anti-angiogenic factor operablyassociated with an expression control sequence that provides forexpression of the anti-angiogenic factor in the manufacture of amedicament for inhibiting growth or metastasis, or both, of a tumor.More particularly, the invention provides for use of a virus vector ofthe invention, e.g., as set out below, in the manufacture of amedicament for inhibiting growth or metastasis, or both, of a tumor.

[0018] Naturally, in addition to the foregoing methods and uses, theinvention provides a novel virus vector comprising a gene encoding ananti-angiogenic factor operably associated with an expression controlsequence. In a preferred embodiment, the virus vector is an adenovirusvector. In a more preferred embodiment, the virus vector is a defectiveadenovirus vector. The virus vectors of the invention can provide a geneencoding any anti-angiogenic factor, as set forth above. For example,the anti-angiogenic factor may comprise a sequence of an amino terminalfragment of urokinase having an EGF-like domain, with the proviso thatthe factor is not urokinase. In a preferred embodiment, theanti-angiogenic factor is an amino terminal fragment of urokinase havingan amino acid sequence of urokinase from amino acid residue 1 to aboutresidue 135. In this embodiment, the urokinase may be murine urokinaseor, preferably, human urokinase.

[0019] The invention further provides a pharmaceutical composition anyof the virus vectors of the invention and a pharmaceutically acceptablecarrier.

[0020] Thus, one object of the invention is to provide gene therapy bydelivery of anti-angiogenic factors for treating tumors.

[0021] Another object of the invention is to provide a viral vector fordelivery of an anti-tumorigenic factor.

[0022] Still another object of the invention is to provide an aminoterminal fragment of urokinase (ATF) by gene therapy for treatment of atumor.

[0023] Further, another object of the invention is to provideangiostatin by gene therapy for treatment of a tumor.

[0024] Yet another object of the invention is to provide angiostatin,particularly kringles 1 to 3 of angiostatin, by gene therapy fortreatment of a tumor.

[0025] These and other objects of the invention are further elaboratedin the following Detailed Description and Examples, and the accompanyingdrawings.

DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1. Molecular characterization of virus AdmATF. Panel A:Structure of AdmATF and AdCO1. The Ad5 chromosome is 36 kb long andbordered by inverted terminal repeats. Y refers to the encapsidationsignal. Both viruses are defective for growth because they lack the Ad5E1 genes. They also carry a 1.9 kb XbaI deletion within region E3. Aschematic representation of the mATF expression cassette of virus AdmATFis indicated under the Ad5 chromosome (not drawn to scale). For a reviewon adenoviral vectors see (38). Panel B: analysis of mATF expression.MDA-MB-231 cells were infected for 24 hr by AdCO1 (lane 2) or AdmATF(lane 3), or mock-infected (lane 1), and total poly(A+) RNAs weresubmitted to northern blot analysis. The ATF-encoding RNA (0.5 kb) isindicated (arrow). A 1.7 kb molecule is also detected (asterisk), a sizein agreement with the utilization of the polyadenylation signal from theadenovirus pIX gene. Panel C: analysis of ATF secretion by 293-infectedcells. The culture media of mock-infected cells (lane 1), or infectedwith AdCO1 (lane 2) or AdmATF (lane 3) were submitted to a western blotanalysis with a polyclonal anti-mouse uPA antibody.

[0027]FIG. 2. Functional characterization of virus AdmATF. Panel A: Theculture medium of AdmATF-infected cells inhibits plasmin conversion atthe surface of LLC cells (see Methods section of the Example). 293refers to the supernatant of non infected cells. Panel B: Infection ofLLC cells with AdmATF (right panel) specifically inhibits cellinvasiveness as compared to that of LLC cells infected with AdCO1 (leftpanel). The 1.2 mm pores of the membranes are visible.

[0028]FIG. 3. Intratumoral injection of AdmATF inhibits LLC tumor growthin syngeneic mice. Tumor cells (2×10⁶ cells) were subcutaneouslyinjected into C57BL/6 mice. After 6 days, the animals received anintratumoral injection of PBS, or 10⁹ PFU of AdCO1 or AdmATF and tumorgrowth was monitored. The mean values are represented with theirstandard variations (n=10). Statistics were done with the Student test.

[0029]FIG. 4. Intratumoral injection of AdmATF inhibits LLC tumorvascularization. Panel A: a representative tumor from the AdCO 1-treated(left) and AdmATF-treated groups extracted at day 10 p.i. is shown. Arepresentative tumor extracted at day 20 p.i. is shown in panel B(injection with AdCO1) and panel C (injection with AdmATF). Allphotographs were taken at the same magnification. Note that theAdmATF-injected tumors are much smaller that their AdCO1-injectedcontrols, especially at the latest time p.i. (compare panels B and C).

[0030]FIG. 5. Intratumoral injection of AdmATF inhibits MDA-MB-231 tumorgrowth in nude mice. Tumors were implanted by subcutaneous injection of3×10⁶ MDA-MB-231 cells. At day 11 post implantation, the mice receivedan intratumoral injection of PBS, or 10⁹ PFU of AdmATF or AdCO1, and thetumor growth was monitored. The mean values are represented with theirstandard variations.

[0031]FIG. 6. Intratumoral injection of AdmATF inhibits intratumoral andperitumoral angiogenesis. Panels A and B: vWF immunostaining of tumorsections. Paraffin embedded MDA-MB-231 tumor sections prepared from theAdCO1— (A) and AdmATF-treated groups (B) were revealed with a polyclonalanti-vWF serum at day 52 p.i. Panels C and D: Macroscopic evaluation ofperitumoral vascularization within the skin of tumors injected withAdCO1(C) or AdmATF (D) at day 20 p.i.

[0032]FIG. 7. (A) Recombinant adenoviruses. The Ad5 genome is a 36kb-long chromosome. Viruses AdK3 and AdCO1 were derived by a lethaldeletion of the E1 genes (nucleotide position 382 to 3446); they alsocarry a non-lethal 1.9 kb XbaI deletion within region E3 (for a reviewsee [37]). The angiostatin expression cassette is shown under the Ad5chromosome. The plasminogen secretion signal is represented by ablackened box; +1 refers to the CMV-driven transcription start; AATAAArefers to the SV40 late polyadenylation signal. (B) Analysis ofangiostatin secretion from infected-cells. 100 ng of human Plg (lane 1),culture medium from HMEC-1 infected with AdK3 (lane 2) or AdCO1 (lane3), C6 infected with AdK3 (lane 4) or AdCO1 (lane 5), and fromMDA-MB-231 infected with AdK3 (lane 6) or AdCO1 (lane 7) were submittedto Western blot analysis. (C) Immuno-detection of angiostatin within C6tumor extracts; Tumors were established in nude mice and received 10⁹PFU of AdCO1 (lane 1) or AdK3 (lane 2) and Western blot analysis wasperformed 10 days p.i. The signal corresponding to angiostatin (36-38kDa) and Plg (92 kDa) are indicated (arrow and asterisk respectively).

[0033]FIG. 8. (A) Inhibition of endothelial cell proliferation. C6(panel 1), MDA-MB-231 (panel 2) and HMEC-1 (panel 3) were injected withAdK3 (♦) or Ad-CO1 (D). HMEC-1 cells (panel 4) cultured with thesupernatant from AdK3-(♦) or AdCO1-infected C6 glioma cells (□). (B)Detection of MPM-2 phosphoepitope in HMEC-1 cells. Mock-infected cells(lane 1), AdCO1-infected cells (lane 2), and AdK3-infected cells (lane3). (C) MPM-2 epitope were detected in HMEC-1 infected with AdCO1(panel 1) or AdK3 (panel 2) by indirect immunostaining and DNA contentby propidium iodide staining, and quantified by flow cytometry (seeMethods). A Student's t-test was used for statistical analysis.

[0034]FIG. 9. AdK3 inhibits tumor growth. C6 glioma (panel A) andMDA-MB-231 carcinoma (panel B) were subcutaneously implanted intoathymic mice (see Methods). When the tumor had reached a volume of 20mm³ (day 0), mice received an intratumoral injection of PBS (□), or 10⁹PFU or AdK3 (•) or AdCO1 (♦). Mean values are represented with theirstandard deviations.

[0035]FIG. 10. AdK3 inhibits C6 tumor growth and angiogenesis. Tumorsfrom AdCO1-treated (panel A) and AdK3-treated groups (panel B) are shown10 days p.i. The extent of vascularization at the periphery of arepresentative tumor injected with AdCO1 (panel C) or AdK3 (panel -D) isshown at day 5 p.i. Paraffin-embedded C6 sections from an AdCO1-injected(panel E) or an AdK3-injected tumor (panel F) were submitted tovWF-immunostaining at day 10 p.i. The proportion of apoptotic cells wasdetected by the TUNEL method within sections from an AdCO1-injected(panel G) or an AdK3-injected tumor (panel H). The same magnificationwas used for AdCO1- and AdK3-injected tumors.

[0036]FIG. 11. Dose dependent effect of AdK3. C6 cells were infected invitro, 24 hours with AdCO1 (panel A) or Ad3K (panel B) and mixed with aratio of 1 (□), 1:2 (♦) and 1:4 (•) with non-infected C6 cells, prior toC6 cells implantation into athymic mice. Tumor volumes were measuredduring 20 days. Mean values are represented with their standarddeviations.

DETAILED DESCRIPTION OF THE INVENTION

[0037] As disclosed above, the present invention is directed to methodsand vectors for gene therapy of tumors. The methods and vectors of theinvention inhibit tumor growth or tumor metastasis, or both. Thesemethods and vectors act by inhibiting angiogenesis of the tumor to anunexpectedly advantageous degree.

[0038] The invention is based, in part, on experiments involving genetherapy delivery of the amino terminal fragment of urokinase (ATF) andangiostatin. ATF is an antagonist of urokinase (uPA) binding to its cellsurface receptor (uPAR), and an inhibitor of endothelial cell migration.To assess the importance of the uPA/uPAR interaction for tumor growthand dissemination, a defective adenovirus expressing murine ATF from theCMV promoter (AdmATF) was constructed. A single intratumoral injectionof AdmATF inhibited growth of pre-established tumors in two differentmurine models, and delayed tumor dissemination. These effects werecorrelated with a remarkable inhibition of neovascularization within,and at the immediate vicinity of, the injection site. The magnitude ofthis effect was particularly remarkable in the ability of murine ATF toinhibit angiogenesis of a human-derived tumor. In a specific example, adefective adenovirus that expresses the N-terminal fragment (aa 1-333)from human Plg, including the pre-activation peptide and kringles 1 to 3[47] was constructed (AdK3) and its in vitro and in vivo activity indifferent murine tumor models was assessed. The AdK3 vector inhibitedtumor growth, tumor angiogenesis, and tumorigenesis, and induced tumorcell apoptosis.

[0039] Intratumoral adenovirus-mediated delivery of antagonist displayspotent antitumoral properties by targeting angiogenesis.

Definitions

[0040] The following defined terms are used throughout the presentspecification, and should be helpful in understanding the scope andpractice of the present invention.

[0041] In a specific embodiment, the term “about” or “approximately”means within 20%, preferably within 10%, and more preferably within 5%of a given value or range.

[0042] An “anti-angiogenic” factor is a molecule that inhibitsangiogenesis, particularly by blocking endothelial cell migration. Suchfactors include fragments of angiogenic proteins that are inhibitory(such as the ATF of urokinase), angiogenesis inhibitory factors, such asangiostatin and endostatin; and soluble receptors of angiogenic factors,such as the urokinase receptor or FGF/VEGF receptor. The term“angiostatin”, which is derived from the amino-terminal fragment ofplasinogen, includes the anti-angiogenic fragment of angiostatin havingkringles 1 to 3. Generally, an anti-angiogenic factor for use in theinvention is a protein or polypeptide encoded by a gene transfected intotumors using the vectors of the invention.

[0043] A “variant” of a polypeptide or protein is any analogue,fragment, derivative, or mutant which is derived from a polypeptide orprotein and which retains at least one biological property of thepolypeptide or protein. Different variants of the polypeptide or proteinmay exist in nature. These variants may be allelic variationscharacterized by differences in the nucleotide sequences of thestructural gene coding for the protein, or may involve differentialsplicing or post-translational modification. The skilled artisan canproduce variants having single or multiple amino acid substitutions,deletions, additions, or replacements. These variants may include, interalia: (a) variants in which one or more amino acid residues aresubstituted with conservative or non-conservative amino acids, (b)variants in which one or more amino acids are added to the polypeptideor protein, (c) variants in which one or more of the amino acidsincludes a substituent group, and (d) variants in which the polypeptideor protein is fused with another polypeptide such as serum albumin. Thetechniques for obtaining these variants, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to persons having ordinary skill in the art.

[0044] If such allelic variations, analogues, fragments, derivatives,mutants, and modifications, including alternative mRNA splicing formsand alternative post-translational modification forms result inderivatives of the polypeptide which retain any of the biologicalproperties of the polypeptide, they are intended to be included withinthe scope of this invention.

General Molecular Biology

[0045] In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sam brook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

[0046] Therefore, if appearing herein, the following terms shall havethe definitions set out below.

[0047] A “vector” is any means for the transfer of a nucleic acidaccording to the invention into a host cell. The term “vector” includesboth viral and nonviral means for introducing the nucleic acid into acell in vitro, ex vivo or in vivo. Non-viral vectors include plasmids,liposomes, electrically charged lipids (cytofectins), DNA-proteincomplexes, and biopolymers. Viral vectors include retrovirus,adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex,Epstein-Barr and adenovirus vectors, as set forth in greater detailbelow. In addition to a nucleic acid according to the invention, avector may also contain one or more regulatory regions, and/orselectable markers useful in selecting, measuring, and monitoringnucleic acid transfer results (transfer to which tissues, duration ofexpression, etc.).

[0048] “Regulatory region” means a nucleic acid sequence which regulatesthe expression of a second nucleic acid sequence. A regulatory regionmay include sequences which are naturally responsible for expressing aparticular nucleic acid (a homologous region) or may include sequencesof a different origin (responsible for expressing different proteins oreven synthetic proteins). In particular, the sequences can be sequencesof eukaryotic or viral genes or derived sequences which stimulate orrepress transcription of a gene in a specific or non-specific manner andin an inducible or non-inducible manner. Regulatory regions includeorigins of replication, RNA splice sites, enhancers, transcriptionaltermination sequences, signal sequences which direct the polypeptideinto the secretory pathways of the target cell, and promoters.

[0049] A regulatory region from a “heterologous source” is a regulatoryregion which is not naturally associated with the expressed nucleicacid. Included among the heterologous regulatory regions are regulatoryregions from a different species, regulatory regions from a differentgene, hybrid regulatory sequences, and regulatory sequences which do notoccur in nature, but which are designed by one having ordinary skill inthe art.

[0050] A “cassette” refers to a segment of DNA that can be inserted intoa vector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

[0051] A cell has been “transfected” by exogenous or heterologous DNAwhen such DNA has been introduced inside the cell. A cell has been“transformed” or “transduced” by exogenous or heterologous DNA when thetransfected DNA effects a phenotypic change.

[0052] “Heterologous” DNA refers to DNA not naturally located in thecell, or in a chromosomal site of the cell. Preferably, the heterologousDNA includes a gene foreign to the cell.

[0053] A “nucleic acid” is a polymeric compound comprised of covalentlylinked subunits called nucleotides. Nucleic acid includespolyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both ofwhich may be single-stranded or double-stranded. DNA includes cDNA,genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence ofnucleotides or nucleic acid sequence that encodes a protein is calledthe sense sequence. A “recombinant DNA molecule” is a DNA molecule thathas undergone a molecular biological manipulation.

[0054] A DNA “coding sequence” is a double-stranded DNA sequence whichis transcribed and translated into a polypeptide in a cell in vitro orin vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxyl) terminus. A polyadenylation signal and transcriptiontermination sequence will usually be located 3′ to the coding sequence.

[0055] Transcriptional and translational control sequences are DNAregulatory sequences, such as promoters, enhancers, terminators, and thelike, that provide for the expression of a coding sequence in a hostcell. In eukaryotic cells, polyadenylation signals are controlsequences.

[0056] A “promoter sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

[0057] A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then optionallytrans-RNA spliced and translated into the protein encoded by the codingsequence.

[0058] A “signal sequence” is included at the beginning of the codingsequence of a protein to be expressed on the surface of a cell. Thissequence encodes a signal peptide, N-terminal to the mature polypeptide,that directs the host cell to translocate the polypeptide. The term“translocation signal sequence” is used herein to refer to this sort ofsignal sequence. Translocation signal sequences can be found associatedwith a variety of proteins native to eukaryotes and prokaryotes, and areoften functional in both types of organisms.

[0059] The term “corresponding to” is used herein to refer similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

[0060] The various aspects of the invention will be set forth in greaterdetail in the following sections, directed to suitable gene therapyvectors and promoters, anti-angiogenic proteins, and therapeuticstrategies. This organization into various sections is intended tofacilitate understanding of the invention, and is in no way intended tobe limiting thereof.

Gene Therapy Vectors

[0061] As discussed above, a “vector” is any means for the transfer of anucleic acid according to the invention into a host cell. Preferredvectors are viral vectors, such as retroviruses, herpes viruses,adenoviruses and adeno-associated viruses. Thus, a gene or nucleic acidsequence encoding an anti-angiogenic protein or polypeptide domainfragment thereof is introduced in vivo, ex vivo, or in vitro using aviral vector or through direct introduction of DNA. Expression intargeted tissues can be effected by targeting the transgenic vector tospecific cells, such as with a viral vector or a receptor ligand, or byusing a tissue-specific promoter, or both.

[0062] Viral vectors commonly used for in vivo or ex vivo targeting andtherapy procedures are DNA-based vectors and retroviral vectors. Methodsfor constructing and using viral vectors are known in the art [see,e.g., Miller and Rosman, BioTechniques 7:980-990 (1992)]. Preferably,the viral vectors are replication defective, that is, they are unable toreplicate autonomously in the target cell. In general, the genome of thereplication defective viral vectors which are used within the scope ofthe present invention lack at least one region which is necessary forthe replication of the virus in the infected cell. These regions caneither be eliminated (in whole or in part), be rendered non-functionalby any technique known to a person skilled in the art. These techniquesinclude the total removal, substitution (by other sequences, inparticular by the inserted nucleic acid), partial deletion or additionof one or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (on the isolated DNA) or in situ,using the techniques of genetic manipulation or by treatment withmutagenic agents. Preferably, the replication defective virus retainsthe sequences of its genome which are necessary for encapsulating theviral particles.

[0063] DNA viral vectors include an attenuated or defective DNA virus,such as but not limited to herpes simplex virus (HSV), papillomavirus,Epstein-Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt etal., Molec. Cell. Neurosci. 2:320-330 (1991)], defective herpes virusvector lacking a glyco-protein L gene [Patent Publication RD 371005 A],or other defective herpes virus vectors [International PatentPublication No. WO 94/21807, published Sep. 29, 1994; InternationalPatent Publication No. WO 92/05263, published Apr. 2, 1994]; anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. [J. Clin. Invest. 90:626-630 (1992); seealso La Salle et al., Science 259:988-990 (1993)]; and a defectiveadeno-associated virus vector [Samulski et al., J. Virol. 61:3096-3101(1987); Samulski et al., J. Virol. 63:3822-3828 (1989); Lebkowski etal., Mol. Cell. Biol. 8:3988-3996 (1988)].

[0064] Preferably, for in vivo administration, an appropriateimmunosuppressive treatment is employed in conjunction with the viralvector, e.g., adenovirus vector, to avoid immuno-deactivation of theviral vector and transfected cells. For example, immunosuppressivecytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), oranti-CD4 antibody, can be administered to block humoral or cellularimmune responses to the viral vectors [see, e.g., Wilson, NatureMedicine (1995)]. In addition, it is advantageous to employ a viralvector that is engineered to express a minimal number of antigens.

[0065] Adenovirus vectors. In a preferred embodiment, the vector is anadenovirus vector. As shown in the Examples, defective adenovirusvectors have shown themselves to be particularly effective for deliveryof the angiogenesis inhibitors ATF and angiostatin, as shown by theunexpectedly efficient effects of inhibiting tumor growth andtumorigenesis. Adenoviruses are eukaryotic DNA viruses that can bemodified to efficiently deliver a nucleic acid of the invention to avariety of cell types. Various serotypes of adenovirus exist. Of theseserotypes, preference is given, within the scope of the presentinvention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5)or adenoviruses of animal origin (see WO94/26914). Those adenoviruses ofanimal origin which can be used within the scope of the presentinvention include adenoviruses of canine, bovine, murine (example: Mav1,Beard et al., Virology 75 (1990) 81), ovine, porcine, avian, and simian(example: SAV) origin. Preferably, the adenovirus of animal origin is acanine adenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan orA26/61 strain (ATCC VR-800), for example).

[0066] Preferably, the replication defective adenoviral vectors of theinvention comprise the ITRs, an encapsidation sequence and the nucleicacid of interest. Still more preferably, at least the E1 region of theadenoviral vector is non-functional. The deletion in the E1 regionpreferably extends from nucleotides 455 to 3329 in the sequence of theAdS adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3Afragment). Other regions may also be modified, in particular the E3region (WO95/02697), the E2 region (WO94/28938), the E4 region(WO94/28152, WO94/12649 and WO95/02697), or in any of the late genesL1-L5.

[0067] In a preferred embodiment, the adenoviral vector has a deletionin the E1 region (Ad 1.0). Examples of E1-deleted adenoviruses aredisclosed in EP 185,573, the contents of which are incorporated hereinby reference. In another preferred embodiment, the adenoviral vector hasa deletion in the E1 and E4 regions (Ad 3.0). Examples of E1/E4-deletedadenoviruses are disclosed in WO95/02697 and WO96/22378, the contents ofwhich are incorporated herein by reference. In still another preferredembodiment, the adenoviral vector has a deletion in the E1 region intowhich the E4 region and the nucleic acid sequence are inserted (see FR9413355, the contents of which are incorporated herein by reference).

[0068] The replication defective recombinant adenoviruses according tothe invention can be prepared by any technique known to the personskilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573;Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared byhomologous recombination between an adenovirus and a plasmid whichcarries, inter alia, the DNA sequence of interest. The homologousrecombination is effected following cotransfection of the saidadenovirus and plasmid into an appropriate cell line. The cell linewhich is employed should preferably (i) be transformable by the saidelements, and (ii) contain the sequences which are able to complementthe part of the genome of the replication defective adenovirus,preferably in integrated form in order to avoid the risks ofrecombination. Examples of cell lines which may be used are the humanembryonic kidney cell line 293 (Graham et al., J. Gen. Virol. 36 (1977)59) which contains the left-hand portion of the genome of an AdSadenovirus (12%) integrated into its genome, and cell lines which areable to complement the E1 and E4 functions, as described in applicationsWO94/26914 and WO95/02697. Recombinant adenoviruses are recovered andpurified using standard molecular biological techniques, which are wellknown to one of ordinary skill in the art.

[0069] Adeno-associated viruses. The adeno-associated viruses (AAV) areDNA viruses of relatively small size which can integrate, in a stableand site-specific manner, into the genome of the cells which theyinfect. They are able to infect a wide spectrum of cells withoutinducing any effects on cellular growth, morphology or differentiation,and they do not appear to be involved in human pathologies. The AAVgenome has been cloned, sequenced and characterized. It encompassesapproximately 4700 bases and contains an inverted terminal repeat (ITR)region of approximately 145 bases at each end, which serves as an originof replication for the virus. The remainder of the genome is dividedinto two essential regions which carry the encapsidation functions: theleft-hand part of the genome, which contains the rep gene involved inviral replication and expression of the viral genes; and the right-handpart of the genome, which contains the cap gene encoding the capsidproteins of the virus.

[0070] The use of vectors derived from the AAVs for transferring genesin vitro and in vivo has been described (see WO 91/18088; WO 93/09239;U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488 528). Thesepublications describe various AAV-derived constructs in which the repand/or cap genes are deleted and replaced by a gene of interest, and theuse of these constructs for transferring the said gene of interest invitro (into cultured cells) or in vivo, (directly into an organism). Thereplication defective recombinant AAVs according to the invention can beprepared by cotransfecting a plasmid containing the nucleic acidsequence of interest flanked by two AAV inverted terminal repeat (ITR)regions, and a plasmid carrying the AAV encapsidation genes (rep and capgenes), into a cell line which is infected with a human helper virus(for example an adenovirus). The AAV recombinants which are produced arethen purified by standard techniques.

[0071] The invention also relates, therefore, to an AAV-derivedrecombinant virus whose genome encompasses a sequence encoding a nucleicacid encoding an anti-angiogenic factor flanked by the AAV ITRs. Theinvention also relates to a plasmid encompassing a sequence encoding anucleic acid encoding an anti-angiogenic factor flanked by two ITRs froman AAV. Such a plasmid can be used as it is for transferring the nucleicacid sequence, with the plasmid, where appropriate, being incorporatedinto a liposomal vector (pseudo-virus).

[0072] Retrovirus vectors. In another embodiment the gene can beintroduced in a retroviral vector, e.g., as described in Anderson etal., U.S. Pat. No. 5,399,346; Mann et al., 1983, Cell 33:153; Temin etal., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;Markowitz et al., 1988, J. Virol. 62:1120; Temin et al., U.S. Pat. No.5,124,263; EP453242, EP178220; Bernstein et al. Genet. Eng. 7 (1985)235; McCormick, BioTechnology 3 (1985) 689; International PatentPublication No. WO 95/07358, published Mar. 16, 1995, by Dougherty etal.; and Kuo et al., 1993, Blood 82:845. The retroviruses areintegrating viruses which infect dividing cells. The retrovirus genomeincludes two LTRs, an encapsidation sequence and three coding regions(gag, pol and env). In recombinant retroviral vectors, the gag, pol andenv genes are generally deleted, in whole or in part, and replaced witha heterologous nucleic acid sequence of interest. These vectors can beconstructed from different types of retrovirus, such as, HIV, MoMuLV(“murine Moloney leukaemia virus” MSV (“murine Moloney sarcoma virus”),HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Roussarcoma virus”) and Friend virus. Defective retroviral vectors aredisclosed in WO95/02697.

[0073] In general, in order to construct recombinant retrovirusescontaining a nucleic acid sequence, a plasmid is constructed whichcontains the LTRs, the encapsidation sequence and the coding sequence.This construct is used to transfect a packaging cell line, which cellline is able to supply in trans the retroviral functions which aredeficient in the plasmid. In general, the packaging cell lines are thusable to express the gag, pol and env genes. Such packaging cell lineshave been described in the prior art, in particular the cell line PA317(U.S. Pat. No. 4,861,719); the PsiCRIP cell line (WO90/02806) and theGP+envAm-12 cell line (WO89/07150). In addition, the recombinantretroviral vectors can contain modifications within the LTRs forsuppressing transcriptional activity as well as extensive encapsidationsequences which may include a part of the gag gene (Bender et al., J.Virol. 61 (1987) 1639). Recombinant retroviral vectors are purified bystandard techniques known to those having ordinary skill in the art.

[0074] Retroviral vectors can be constructed to function as infectiousparticles or to undergo a single round of transfection. In the formercase, the virus is modified to retain all of its genes except for thoseresponsible for oncogenic transformation properties, and to express theheterologous gene. Non-infectious viral vectors are prepared to destroythe viral packaging signal, but retain the structural genes required topackage the co-introduced virus engineered to contain the heterologousgene and the packaging signals. Thus, the viral particles that areproduced are not capable of producing additional virus.

[0075] Targeted gene delivery is described in International PatentPublication WO 95/28494, published October 1995.

[0076] Non-viral Vectors. Alternatively, the vector can be introduced invivo as nucleic acid free of transfecting excipients, or withtransfection facilitating agents, e.g., lipofection. For the pastdecade, there has been increasing use of liposomes for encapsulation andtransfection of nucleic acids in vitro. Synthetic cationic lipidsdesigned to limit the difficulties and dangers encountered with liposomemediated transfection can be used to prepare liposomes for in vivotransfection of a gene encoding a marker [Felgner, et. al., Proc. Natl.Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey, et al., Proc. Natl.Acad. Sci. U.S.A. 85:8027-8031 (1988); Ulmer et al., Science259:1745-1748 (1993)]. The use of cationic lipids may promoteencapsulation of negatively charged nucleic acids, and also promotefusion with negatively charged cell membranes [Felgner and Ringold,Science 337:387-388 (1989)]. Particularly useful lipid compounds andcompositions for transfer of nucleic acids are described inInternational Patent Publications WO95/18863 and WO96/17823, and in U.S.Pat. No. 5,459,127. The use of lipofection to introduce exogenous genesinto the specific organs in vivo has certain practical advantages.Molecular targeting of liposomes to specific cells represents one areaof benefit. It is clear that directing transfection to particular celltypes would be particularly advantageous in a tissue with cellularheterogeneity, such as pancreas, liver, kidney, and the brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting [see Mackey, et. al., supra]. Targeted peptides, e.g.,hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

[0077] Other molecules are also useful for facilitating transfection ofa nucleic acid in vivo, such as a cationic oligopeptide (e.g.,International Patent Publication WO95/21931), peptides derived from DNAbinding proteins (e.g., International Patent Publication WO96/25508), ora cationic polymer (e.g., International Patent Publication WO95/21931).

[0078] It is also possible to introduce the vector in vivo as a nakedDNA plasmid. Naked DNA vectors for gene therapy can be introduced intothe desired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter [see, e.g., Wu et al., J. Biol. Chem. 267:963-967(1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut etal., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990;Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991)].Receptor-mediated DNA delivery approaches can also be sued [Curiel etal., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J. Biol. Chem.262:4429-4432 (1987)].

[0079] The nucleic acid can also be administered as a naked DNA. Methodsfor formulating and administering naked DNA to mammalian muscle tissueare disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466, the contents ofwhich are incorporated herein by reference.

[0080] Regulatory Regions. Expression of an anti-angiogenic factor froma vector of the invention may be controlled by any regulatory region,i.e., promoter/enhancer element known in the art, but these regulatoryelements must be functional in the host target tumor selected forexpression.

[0081] The regulatory regions may comprise a promoter region forfunctional transcription in the tumor, as well as a region situated in3′ of the gene of interest, and which specifies a signal for terminationof transcription and a polyadenylation site. All these elementsconstitute an expression cassette.

[0082] Promoters that may be used in the present invention include bothconstitutive promoters and regulated (inducible) promoters. The promotermay be naturally responsible for the expression of the nucleic acid. Itmay also be from a heterologous source. In particular, it may bepromoter sequences of eukaryotic or viral genes. For example, it may bepromoter sequences derived from the genome of the cell which it isdesired to infect. Likewise, it may be promoter sequences derived fromthe genome of a virus, including the adenovirus used. In this regard,there may be mentioned, for example, the promoters of the EIA, MLP, CMVand RSV genes and the like.

[0083] In addition, the promoter may be modified by addition ofactivating or regulatory sequences or sequences allowing atissue-specific or predominant expression (enolase and GFAP promotersand the like). Moreover, when the nucleic acid does not contain promotersequences, it may be inserted, such as into the virus genome downstreamof such a sequence.

[0084] Some promoters useful for practice of this invention areubiquitous promoters (e.g., HPRT, vimentin, actin, tubulin),intermediate filament promoters (e.g., desmin, neurofilaments, keratin,GFAP), therapeutic gene promoters (e.g., MDR type, CFTR, factor VIII),tissue-specific promoters (e.g., actin promoter in smooth muscle cells),promoters which are preferentially activated in dividing cells,promoters which respond to a stimulus (e.g., steroid hormone receptor,retinoic acid receptor), tetracycline-regulated transcriptionalmodulators, cytomegalovirus immediate-early, retroviral LTR,metallothionein, SV-40, E1a, and MLP promoters. Tetracycline-regulatedtranscriptional modulators and CMV promoters are described in WO96/01313, U.S. Pat. Nos. 5,168,062 and 5,385,839, the contents of whichare incorporated herein by reference.

[0085] Thus, the promoters which may be used to control gene expressioninclude, but are not limited to, the cytomegalovirus (CMV) promoter, theSV40 early promoter region (Benoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the b-lactamase promoter (Villa-Kamaroff, et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also“Useful proteins from recombinant bacteria” in Scientific American,1980, 242:74-94; promoter elements from yeast or other fungi such as theGal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter; andthe animal transcriptional control regions, which exhibit tissuespecificity and have been utilized in transgenic animals: elastase Igene control region which is active in pancreatic acinar cells (Swift etal., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring HarborSymp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515);insulin gene control region which is active in pancreatic beta cells(Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control regionwhich is active in lymphoid cells (Grosschedl et al., 1984, Cell38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al.,1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus controlregion which is active in testicular, breast, lymphoid and mast cells(Leder et al., 1986, Cell 45:485-495), albumin gene control region whichis active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science235:53-58), alpha 1-antitrypsin gene control region which is active inthe liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globingene control region which is active in myeloid cells (Mogram et al.,1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelinbasic protein gene control region which is active in oligodendrocytecells in the brain (Readhead et al., 1987, Cell 48:703-712), myosinlight chain-2 gene control region which is active in skeletal muscle(Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science 234:1372-1378).

Genes Encoding Anti-Angiogenic Proteins

[0086] The vectors of the invention can be used to deliver a geneencoding an anti-angiogenic protein into a tumor in accordance with theinvention. In a preferred embodiment, the anti-angiogenic factor is theamino terminal fragment (ATF) of urokinase, containing the EGF-likedomain. Such fragment corresponds to amino acid residues about 1 toabout 135 of ATF.

[0087] In another embodiment, ATF may be provided as a fusion protein,e.g., with immunoglobulin or human serum albumin [WO93/15199], which isspecifically incorporated herein by reference in its entirety.

[0088] An effective ATF for use in the invention can be derived from anyurokinase, such as murine urokinase, although human urokinase ATF ispreferred. In addition, the invention contemplates administration of anon-human urokinase ATF modified by substitution of specific amino acidresidues with the corresponding residues from human ATF. For example,murine ATF can be modified at one or more, and preferably all, aminoacid residues as follows: tyrosine-23 to asparagine; arginine-28 toasparagine; arginine-30 to histidine; and arginine-31 to tryptophan.Thus, urokinase ATF from any source can be humanized. This is easilyaccomplished by modifying the coding sequence using routine molecularbiological techniques.

[0089] Genes encoding other anti-angiogenesis protein can also be usedaccording to the invention. Such genes include, but are not limited to,genes encoding angiostatin [O'Reilly et al., Cell 79:315-328 (1994);WO95/29242; U.S. Pat. No. 5,639,725], including angiostatin comprisingkringles 1 to 3; tissue inhibition of metalloproteinase [Johnson et al.,J. Cell. Physiol. 160:194-202 (1994)]; inhibitors of FGF or VEGF; andendostatin [WO97/15666]. In a preferred embodiment, the anti-angiogenicfactor is angiostatin, particularly kringles 1 to 3 of angiostatin. In aparticularly preferred embodiment, the anti-angiogenic factor is theamino-terminal fragment of plasminogen (Plg) having an amino acidsequence of plasminogen from about amino acid residue I to about residue333. In another preferred embodiment, the amino terminal fragment ofplasminogen/angiostatin is human plasminogen (angiostatin).

[0090] In another embodiment, the invention provides for administrationof genes encoding soluble forms of receptors for angiogenic factors,including but not limited to soluble VGF/VEGF receptor, and solubleurokinase receptor [Wilhem et al., FEBS Letters 337:131-134 (1994)].

[0091] In general, any gene encoding a protein or soluble receptor thatantagonizes endothelial cell activation and migration, which is involvedin angiogenesis, can be employed in the gene therapy vectors and methodsof the invention. Notwithstanding, it is particularly noteworthy thatgene therapy delivery of ATF or angiostatin is especially effective inthis regard, for reasons pointed out above and exemplified below.

[0092] A gene encoding an anti-angiogenic factor, whether genomic DNA orcDNA, can be isolated from any source, particularly from a human cDNA orgenomic library. Methods for obtaining such genes are well known in theart, as described above [see, e.g., Sambrook et al., 1989, supra].

[0093] Due to the degeneracy of nucleotide coding sequences, othernucleic acid sequences which encode substantially the same amino acidsequence as an anti-angiogenic factor gene may be used in the practiceof the present invention and these are contemplated as falling withinthe scope of the claimed invention. These include but are not limited toallelic genes, homologous genes from other species, and nucleotidesequences comprising all or portions of anti-angiogenic factor geneswhich are altered by the substitution of different codons that encodethe same amino acid residue within the sequence, thus producing a silentchange. Likewise, the anti-angiogenic factor derivatives of theinvention include, but are not limited to, those containing, as aprimary amino acid sequence, all or part of the amino acid sequence ofan anti-angiogenic factor protein including altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a conservative amino acid substitution.For example, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. Amino acidscontaining aromatic ring structures are phenylalanine, tryptophan, andtyrosine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to affect apparentmolecular weight as determined by polyacrylamide gel electrophoresis, orisoelectric point.

[0094] Particularly preferred substitutions are:

[0095] Lys for Arg and vice versa such that a positive charge may bemaintained;

[0096] Glu for Asp and vice versa such that a negative charge may bemaintained;

[0097] Ser for Thr such that a free —OH can be maintained; and

[0098] Gln for Asn such that a free CONH₂ can be maintained.

[0099] The genes encoding anti-angiogenic factor derivatives and analogsof the invention can be produced by various methods known in the art.The manipulations which result in their production can occur at the geneor protein level. For example, the cloned anti-angiogenic factor genesequence can be modified by any of numerous strategies known in the art(Sambrook et al., 1989, supra). The sequence can be cleaved atappropriate sites with restriction endonuclease(s), followed by furtherenzymatic modification if desired, isolated, and ligated in vitro. Inthe production of the gene encoding a derivative or analog ofanti-angiogenic factor, care should be taken to ensure that the modifiedgene remains within the same translational reading frame as theanti-angiogenic factor gene, uninterrupted by translational stopsignals, in the gene region where the desired activity is encoded.

[0100] Additionally, the anti-angiogenic factor-encoding nucleic acidsequence can be mutated in vitro or in vivo, to create and/or destroytranslation, initiation, and/or termination sequences, or to createvariations in coding regions and/or form new restriction endonucleasesites or destroy preexisting ones, to facilitate further in vitromodification, such as to form a chimeric gene. Preferably, such utationsenhance the functional activity of the mutated anti-angiogenic factorgene product. Any technique for mutagenesis known in the art can beused, including but not limited to, in vitro site-directed mutagenesis(Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551; Zoller andSmith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44:177;Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:710), use ofTAB® linkers (Pharmacia), etc. PCR techniques are preferred for sitedirected mutagenesis (see Higuchi, 1989, “Using PCR to Engineer DNA”, inPCR Technology: Principles and Applications for DNA Amplification, H.Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

Therapeutic Targets and Strategies

[0101] The process according to the present invention enables one totreat tumors. According to the present invention, it is now possible, bya judicious choice of various injections, infusions, direct application,etc., to infect specifically and unilaterally a large number of tumorcells.

[0102] Pharmaceutical Compositions. For their use according to thepresent invention, the vectors, either in the form of a virus vector,nucleic acid-lipid composition, or naked DNA, are preferably combinedwith one or more pharmaceutically acceptable carriers for an injectableformulation. The phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are physiologically tolerableand do not typically produce an allergic or similar untoward reaction,such as gastric upset, dizziness and the like, when administered to ahuman. Preferably, as used herein, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopoeia or other generallyrecognized pharmacopoeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the compound is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussolution saline solutions and aqueous dextrose and glycerol solutionsare preferably employed as carriers, particularly for injectablesolutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. These may be inparticular isotonic, sterile, saline solutions (monosodium or disodiumphosphate, sodium, potassium, calcium or magnesium chloride and the likeor mixtures of such salts), or dry, especially freeze-dried compositionswhich upon addition, depending on the case, of sterilized water orphysiological saline, allow the constitution of injectable solutions.

[0103] The preferred sterile injectable preparations can be a solutionor suspension in a nontoxic parenterally acceptable solvent or diluent.Examples of pharmaceutically acceptable carriers are saline, bufferedsaline, isotonic saline (e.g., monosodium or disodium phosphate, sodium,potassium, calcium or magnesium chloride, or mixtures of such salts),Ringer's solution, dextrose, water, sterile water, glycerol, ethanol,and combinations thereof. 1,3-butanediol and sterile fixed oils areconveniently employed as solvents or suspending media. Any bland fixedoil can be employed including synthetic mono- or di-glycerides. Fattyacids such as oleic acid also find use in the preparation ofinjectables.

[0104] The phrase “therapeutically effective amount” is used herein tomean an amount sufficient to reduce by at least about 15 percent,preferably by at least 50 percent, more preferably by at least 90percent, and most preferably prevent, a clinically significant deficitin the activity, function and response of the host. Alternatively, atherapeutically effective amount is sufficient to cause an improvementin a clinically significant condition in the host.

[0105] The virus doses used for the administration may be adapted as afunction of various parameters, and in particular as a function of thesite (tumor) of administration considered, the number of injections, thegene to be expressed or alternatively the desired duration of treatment.In general, the recombinant adenoviruses according to the invention areformulated and administered in the form of doses of between 10⁴ and 10¹⁴pfu, and preferably 10⁶ to 10¹¹ pfu. The term pfu (plaque forming unit)corresponds to the infectivity of a virus solution, and is determined byinfecting an appropriate cell culture and measuring, generally after 15days, the number of plaques of infected cells. The technique fordetermining the pfu titre of a viral solution are well documented in theliterature.

[0106] In a preferred embodiment, the composition comprises anadenovirus comprising the anti-angiogenic factor gene, e.g., ATF gene(AdATF) or angiostatin (AdK3), in a concentration of about 1×10⁹ pfu/100μl.

[0107] The compositions according to the invention are particularlyuseful for administration to tumors.

[0108] Tumors. The present invention is directed the treatment oftumors, particularly solid tumors. Examples of solid tumors that can betreated according to the invention include sarcomas and carcinomas suchas, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma.

[0109] In another embodiment, dysproliferative changes (such asmetaplasias and dysplasias) are treated or prevented in epithelialtissues such as those in the cervix, esophagus, and lung. Thus, thepresent invention provides for treatment of conditions known orsuspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, 1976, BasicPathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79).Hyperplasia is a form of controlled cell proliferation involving anincrease in cell number in a tissue or organ, without significantalteration in structure or function. As but one example, endometrialhyperplasia often precedes endometrial cancer. Metaplasia is a form ofcontrolled cell growth in which one type of adult or fullydifferentiated cell substitutes for another type of adult cell.Metaplasia can occur in epithelial or connective tissue cells. Atypicalmetaplasia involves a somewhat disorderly metaplastic epithelium.Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia; it is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplastic cells often haveabnormally large, deeply stained nuclei, and exhibit pleomorphism.Dysplasia characteristically occurs where there exists chronicirritation or inflammation, and is often found in the cervix,respiratory passages, oral cavity, and gall bladder. For a review ofsuch disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B.Lippincott Co., Philadelphia.

[0110] The present invention is also directed to treatment ofnon-malignant tumors and other disorders involving inappropriate cell ortissue growth augmented by angiogenesis by administering atherapeutically effective amount of a vector of the invention to thetissue undergoing inappropriate growth. For example, it is contemplatedthat the invention is useful for the treatment of arteriovenous (AV)malformations, particularly in intracranial sites. The invention mayalso be used to treat psoriasis, a dermatologic condition that ischaracterized by inflammation and vascular proliferation; and benignprostatic hypertrophy, a condition associated with inflammation andpossibly vascular proliferation. Treatment of other hyperproliferativedisorders is also contemplated.

[0111] Methods of administration. According to the invention, thepreferred route of administration to a tumor is by direct injection intothe tumor. The tumor can be imaged using any of the techniques availablein the art, such as magnetic resonance imaging or computer-assistedtomography, and the therapeutic composition administered by stereotacticinjection, for example.

[0112] Alternatively, if a tumor target is characterized by a particularantigen, a vector of the invention can be targeted to the antigen asdescribed above, and administered systemically or subsystemically, asappropriate, e.g., intravenously, intraarterioally, intraperitoneally,intraventricularly, etc.

[0113] Combination Therapies. Although the methods of the invention areeffective in inhibiting tumor growth and metastasis, the vectors andmethods of the present invention are advantageously used with othertreatment modalities, including without limitation surgery, radiation,chemotherapy, and other gene therapies.

[0114] For example, the vectors of the invention can be administered incombination with nitric oxide inhibitors, which have vasoconstrictiveactivity and reduce blood flow to the tumor.

[0115] In another embodiment, a vector of the invention can beadministered with a chemotherapeutic such as, though not limited to,taxol, taxotere and other taxoids [e.g., as disclosed in U.S. Pat. Nos.4,857,653; 4,814,470; 4,924,011, 5,290,957; 5,292,921; 5,438,072;5,587,493; European Patent No. 0 253 738; and International PatentPublication Nos. WO91/17976, WO93/00928, WO93/00929, and WO9601815], orother chemotherapeutics, such as cis-platin (and other platinintercalating compounds), etoposide and etoposide phosphate, bleomycin,mitomycin C, CCNU, doxorubicin, daunorubicin, idarubicin, ifosfamide,and the like.

[0116] In still another embodiment, a vector of the invention can beadministered in conjunction with another gene therapy for tumors, suchas but by no means limited to p53 or analogues thereof such as CTS-1[WO97/04092], thymidine kinase (TK), anti-RAS single chain antibodies,interferon-α or interferon-γ, etc., as described above. Any vector forgene therapy can be used in conjunction with the present invention, suchas a viral vector or naked DNA. In a preferred embodiment, a singlevector (virus or DNA) is used to deliver genes coding for both ananti-angiogenesis factor and another tumor therapy gene.

[0117] In another aspect, the present invention provides for regulatedexpression of the anti-angiogenic factor gene in concert with expressionof proteins useful in the context of treatment for proliferativedisorders, such as tumors and cancers, when the heterologous geneencodes a targeting marker or immunomodulatory cytokine that enhancestargeting of the tumor cell by host immune system mechanisms. Examplesof such heterologous genes for immunomodulatory (or immuno-effector)molecules include, but are not limited to, interferon-α, interferon-γ,interferon-β, interferon-ω, interferon-1, tumor necrosis factor-α, tumornecrosis factor-, interleukin-2, interleukin-7, interleukin-12,interleukin-15, B7-1 T cell costimulatory molecule, B7-2 T cellcostimulatory molecule, immune cell adhesion molecule (ICAM)1 T cellcostimulatory molecule, granulocyte colony stimulatory factor,granulocyte-macrophage colony stimulatory factor, and combinationsthereof.

[0118] The present invention will be better understood be reference tothe following Examples, which are provided by way of exemplification andnot by way of limitation.

EXAMPLE 1 Gene Therapy With ATF Inhibits Tumor Growth and Metastasis

[0119] Example 1 demonstrates that expression of the uPA/uPAR antagonistATF (amino terminal fragment of urokinase) inhibits tumor growth andmetastasis. A defective adenovirus expressing murine ATF from the CMVpromoter (AdmATF) was constructed. A single intratumoral injection ofAdmATF inhibited growth of pre-established tumors in two differentmurine models, and delayed tumor dissemination. These effects werecorrelated with a remarkable inhibition of neovascularization within,and at the immediate vicinity of, the injection site. The magnitude ofthis effect was particularly remarkable in the ability of murine ATF toinhibit angiogenesis of a human-derived tumor.

Methods

[0120] Recombinant adenoviruses. AdmATF is an E1-defective recombinantadenovirus that expresses the murine ATF gene from the CMV promoter.Plasmid pDB 1519 16 was used as starting material to introduce a stopcodon after residue 135 of mature uPA. Briefly, the uPA-encodingsequences (including its signal peptide) were isolated, restricted byNsiI, and residues 128 to 135 followed by a stop codon were reintroducedas a synthetic fragment. The ATF open reading frame was then insertedbetween the CMV promoter and the SV40 late polyadenylation signalsequence, generating plasmid pEM8-mATF. This plasmid also carries thefirst 6.3 kb of the AdS genome except that the ATF expression cassettehas been inserted between position 382 and 3446, in place of the Elgenes (FIG. 1A). AdmATF was constructed in 293 cells by homologousrecombination between pEM8-mATF and ClaI-restricted AdRSVbGal DNA 25.Individual viral plaques were isolated onto 293-derived cell monolayersgrown in soft agar, amplified onto fresh 293 cells and viral DNA wasextracted 26. EcORI, EcORV and AvrII+NdeI restriction analyses confirmedthe identity and clonality of the recombinant adenovirus. AdCO1 is adefective control adenovirus that is identical to AdmATF except that itdoes not carry any transgene expression cassette in place of E1a. Bothviruses were propagated in 293, a human embryonic kidney cell line thatconstitutively expresses the E1 genes of AdS 27. Viral stocks wereprepared and titrated as described 25. Unless otherwise stated,MDA-MB-231 cells and Lewis lung carcinoma (LLC) cells were infected at amultiplicity of infection (MOI) of 300 PFU/cell. These infectionconditions were previously shown to translate respectively into 80 and65% of b-galactosidase-expressing cells when virus AdRSVbGal 25 wasused.

[0121] Northern blot analysis. Subconfluent MDA-MB-231 cell cultureswere infected with AdmATF or AdCO1, and total RNA was extracted 24 hrpost-infection (p.i.) by the RNAZOL procedure (Biogentex, Inc), andpolyadenylated RNAs were purified. The samples were run in a 1% formaldehyde agarose gel, and transferred onto Hybond N membranes(Amersham). The membranes were prehybridized with denatured sonicatedsalmon sperm DNA (100 μg/ml) for 1 hr at 42° C. in 10 ml of 50%deionized formamide, 0.2% SDS, 5× Denhardt's solution, and incubatedovernight with a random-primed (³²P)-labeled 1.2 kb XbaI-HindIIIfragment from murine uPA cDNA (16). The membranes were washed twice in2×SSC/0.1% SDS for 1 hr at 50° C., once in 0.1×SSC for 30 min, andexposed to Kodak XAR-5 films for 1 hr at room temperature.

[0122] Western blot analysis. Supernatants from virally-infected cellswere collected 24 hr p.i., run in a 12.5% SDS-polyacrylamide gel (400 μgof protein per lane), prior to transfer onto a nitrocellulose membrane(Schleicher & Schuell). After incubation for 1 hr in blocking buffer,the membranes were incubated for 1 hr with a polyclonal serum raisedagainst murine uPA (Pr. R Lijnen, Leuven, Belgium), then for anadditional hour with a horse-radish peroxidase-conjugated goatanti-rabbit serum (Dako). The membranes were washed three times inPBS-Tween buffer, and incubated with 3-Amino-9-ethyl-Carbazole (AEC) for5 min.

[0123] Inhibition of cell-associated proteolysis. Native uPA was firstdissociated from its cell surface receptor by submitting LLC cellmonolayers to a 3 min acidification in glycine-HCl (pH 3), followed byincubation in 0.5 M HEPES buffer. The cells were then incubated for 20min at 37° C. with the supernatant of AdCO1- and AdmATF-infected 293cells. After 3 washes in PBS/0.1% BSA, the cells were incubated at 37°C. for 20 min with 1 nM of murine uPA (Pr. R. Lijnen, Leuven, Belgium).Unbound uPA was then removed by washing in PBS, and cell-associated uPAwas quantified by adding 0.4 μM of human plasminogen and plasminsubstrate S-2251 (Kabi Vitrum, Sweden).

[0124] In vitro invasion assay. Twenty four hr p.i., LLC cells weredetached with 1 mM EDTA, washed in PBS, and resuspended in FCS-free MDEMmedium supplemented with 0.1% BSA. Invasion assays were carried out in atranswell unit as described (19). Briefly, polycarbonate filters of 1.2μm pore size (Transwell, Costar) were coated with 160 μg Matrigel(Becton Dickinson) and dried. The lower chambers of the Transwell unitswere filled with human fibroblast-conditioned medium containing 10 ng/mlEGF, and the upper chambers were seeded with 3×10⁵ infected cells. After24 hr incubation at 37° C., the number of cells that had reached thelower chamber was determined under a light microscope following stainingwith Giesma.

[0125] Syngeneic tumor model. Lewis lung carcinomas were seriallypassaged onto C57BL/6 syngeneic mice. Briefly, C57BL/6 implantedsubcutaneously with a LLC tumor were sacrificed when the tumor hadreached a volume of 600-1200 mm³. Tumor cells were resuspended in a 0.9%saline solution following filtration through a cotton sieve, and 2×10⁶cells (0.5 ml) were subcutaneously implanted to the dorsa of 6-7weeks-old C57BL/6 female mice. After 5 days, the tumors had reached asize of approximately 20 mm³, and they were injected with 0.2 ml of PBS(n=8), or 109 PFU (0.2 ml) of AdCO1 (n=10) or AdmATF (n=10). The size ofthe primary tumor was measured at day 5, 10 and 15 p.i. At day 16 p.i.,the number of lung metastases was assessed 3 hr after an intraperitonealinjection of 65 mg BrdU. Lung tissues were removed, fixed overnight inacetic formaldehyde acid (AFA), and paraffin sections were incubated 15min in 4N HCl, neutralized and saturated by washing twice for 15 min inPBS/0.5% BSA/0.1% Tween 20 prior to incubation with peroxidase-labeledmouse anti-BrdU monoclonal antibody (Boehringer) for 45 min at 37° C.,and AEC. BrdU-positive foci were quantified under a light microscope ata magnification of 25.

[0126] Athymic murine model. Cultured MDA-MB-231 cells (ATCC HTB 26)were harvested, washed, resuspended in PBS at 1.5×10⁷ cells/ml, and3×10⁶ cells were subcutaneously injected in the dorsa of 6-7 weeks oldnude mice. When the tumors had reached a volume of 15-20 mm³ (i.e.,after 11 days), the animals received an intratumoral injection of 109PFU of AdmATF (n=5) or AdCO1 (n=5), or PBS (n=5), and the size of thetumors was monitored until day 52 p.i., after which the animals weresacrificed and the extent of intratumoral vascularization was assessedas described (28). Briefly, tumor tissues were fixed overnight in AFA,transferred to 100% ethanol, embedded in paraffin and 5 μm thicksections were prepared. After toluene treatment and rehydration, thesections were permeabilized with 2 μg/ml proteinase K at 37° C. for 15min. Endogenous peroxidase activity was quenched by 0.3% H₂O₂ for 15min. The sections were washed with PBS, incubated 15 min in 7.5% BSA,and incubated 30 min with a rabbit polyclonal serum raised against humanvWF (Dako). After two washes in PBS, the sections were incubated withbiotinylated goat anti-rabbit IgG antibodies for 30 min, washed, andincubated with streptavidin-peroxidase for 15 min prior to addition ofAEC. Neovascular hotspots were first identified at low magnification andvWF-positive microvessels were quantified. Meyer's hematoxylin was usedfor counterstaining as described (28).

[0127] To evaluate AdmATF infection on tumor establishment, confluentMDA-MB-231 cells were first infected with AdmATF or AdCO1 at an MOI of50 PFU/cell. The cells were washed 24 hr p.i., resuspended in 120 μlPBS, mixed with 80 μl ice-cold Matrigel, and 1.3×10⁶ cells weresubcutaneously implanted into the dorsa of nude mice. Tumorestablishment and growth were followed until day 51 after implantation.

Results

[0128] Molecular and functional characterization of AdmATF. AdmATF is adefective recombinant adenovirus that expresses murine ATF from the CMVpromoter whereas AdCO1 is an “empty” control adenovirus (FIG. 1A). Invitro studies were first carried out to characterize AdmATF with regardsto its ability to express a functional uPA antagonist followinginfection. ATF gene expression was demonstrated by northern analysis ofpoly(A+) RNAs extracted from MDA-MB-231 cells infected for 24 hr withAdmATF, but not AdCO1 (FIG. 1B). Secretion of ATF by AdmATF-infectedcells was demonstrated for 293, LLC and MDA-MB-231 cells by Western blotanalysis. For example, an ATF-specific polypeptide with a molecularweight corresponding to that of the mature peptide (15.3 kDa) isuniquely detected in the medium from 293 cells infected for 24 hr p.i.with AdmATF (FIG. 1C).

[0129] ATF is a potent antagonist of uPA binding to its cell surfacereceptor (uPAR), and disruption of this complex is known to greatlyinhibit the conversion of inactive plasminogen into plasmin. LLCcell-associated plasmin conversion was thus measured to assess thefunctionality of ATF secreted by AdmATF-infected cells. As aprerequisite, we checked that LLC cells displayed significant levels ofcell-associated uPA activity (data not shown), implying that theysecrete uPA and express uPAR. Plasmin conversion/activity wassignificantly reduced when endogenous uPA had been previously removedfrom the cell surface by a mild acid treatment prior to incubation withthe supernatant of AdmATF-infected 293 cells and addition of 1 nM murineuPA (FIG. 2A).

[0130] The uPA/uPAR complex is also crucial to cell motility. An invitro cell invasion assay was used to confirm the functionality ofAdmATF. LLCs cells were infected with AdmATF or AdCO1, and the number ofcells that had migrated through a matrix-coated membrane was determinedafter 24 hr (FIG. 2B). Quantification of the data demonstrated thatAdmATF infection inhibited LLC invasiveness by 65% (n=5) as compared toAdCO1 control infections.

[0131] Intratumoral injection of AdmATF inhibits tumor growth anddissemination. We first used the Lewis lung carcinoma-C57BV6 syngeneicmodel to evaluate the antitumoral effects associated with a singleintratumoral administration of AdmATF. Five days after subcutaneousimplantation, the tumors were injected with 10⁹ PFU of AdmATF, 10⁹ PFUof AdCO1, or PBS, and tumor growth was monitored until day 15 p.i. Asshown in FIG. 3, an overall inhibition was specifically observed in theAdmATF-treated group. The animals were then sacrificed at day 16 p.i.,and lung metastases were numbered by counting the number ofBrdU-positive foci. Whereas metastases were apparent in all animalsinjected with PBS (n=8), 7 out of 9, and only 3 out of 9 scored positivewithin the AdCO1- and AdmATF-treated groups, respectively. The averagenumber of BrdU-positive foci per lung sections was also reduced in theAdmATF-treated group (2.7) as compared to that in the AdCO1-treated(6.3) and PBS-treated (6.6) groups. A single intratumoral administrationof AdmATF therefore significantly inhibited tumor growth and lungdissemination in this highly aggressive model. In a separate experiment,tumor-bearing animals were infected with AdCO1 or AdmATF, and the tumorsextracted at day 10 and 20 p.i. for macroscopic inspection. WhileAdCO1-injected LLC tumors displayed an intense vascularization at bothtime points, tumors from the AdmATF-treated group displayed onlymarginal vascularization (FIG. 4).

[0132] The antitumoral effects of AdmATF are exerted at the level ofangiogenesis. To specifically evaluate the sole inhibition ofangiogenesis for tumor growth, we studied adenovirus-mediated deliveryof the murine uPA/uPAR antagonist in the human-derived MDA-MB-231 breastcarcinoma model implanted into athymic mice. A direct action of murineATF on the tumor cells should be minimal in this model as murine uPAbinds human uPAR 200-fold less efficiently than murine uPAR. Eleven daysafter subcutaneous tumor cell inoculation, the animals received a singleintratumoral injection of 10⁹ PFU of AdmATF, 10⁹ PFU of AdCO1, or PBS,and tumor growth was monitored until day 52 p.i. While no significanteffect were apparent until day 32 p.i., an arrest of tumor growth thenbecame evident in the AdmATF-infected, but not the AdCO1-infected group(FIG. 5). Mice were sacrificed at day 52 p.i., and intratumoralangiogenesis was assessed by visualization of von Willebrand Factor(vWF)-immunoreactive vessels (FIG. 6A). An average of 4 to 6 vesselswere detected within the sections from the AdmATF-treated tumors ascompared to 18 to 20 in the sections from the AdCO1-injected tumors.Tumors injected with AdmATF also displayed little peripheralneovascularization as compared to their AdCO1-treated counterparts (FIG.6B). When MDA-MB-231 cells were first infected in vitro beforesubcutaneous inoculation in the presence of Matrigel, tumors becameapparent in the AdCO1-treated group as early as 7 dayspost-implantation. A tumor of limited size was apparent in only oneanimal from the AdmATF-treated group (n=5), in sharp contrast to thelarger tumors present in 4 out of 5 animals from the AdCO1-infectedgroup. Again, the tumor that had developed following inoculation ofAdmATF-infected tumor cells was less vascularized than those thatdeveloped following inoculation of AdCO1-infected cells (data notshown).

Discussion

[0133] We have studied the antitumoral effects associated with the localdelivery of the amino-terminal, non-catalytic, fragment of urokinase(ATF), a potent antagonist of urokinase binding to its receptor (uPAR)at the surface of both tumor (19, 20) and endothelial cells (22, 23). Invivo delivery of ATF was achieved by intratumoral administration of adefective adenovirus that expresses a secretable ATF molecule of murineorigin from the CMV promoter (AdmATF). To exclude non-specific cytotoxiceffects consecutive to virus infection (29), an “empty” otherwiseisogenic adenovirus (AdCO1) was used as a control virus throughout thestudy. This is an important control also because recombinantadenoviruses can use the aVb3 integrin for infection (30), a cellsurface receptor somehow involved in tumor growth and angiogenesis (31).

[0134] A single intratumoral injection of AdmATF is efficient inreducing tumor growth (FIG. 3) and delaying dissemination to the lungsin the aggressive LLC-C57BL/6 syngeneic murine model. Murine ATFapparently partly exerted these effects by inhibiting the invasivenessof the tumor cell themselves (FIG. 2B), a result consistent with theinhibition of cell-associated proteolysis following AdmATF infection(FIG. 2A). ATF-based antagonists are also potent inhibitors ofendothelial cells motility (22, 23), suggesting that inhibition of tumorangiogenesis may have also contributed to the effects observed in thismodel. Indeed, LLC tumors injected with AdmATF displayed very littlevascularization as compared to AdCO1-infected control tumors (FIG. 4).That specific AdmATF-mediated tumor growth inhibition became evident atlate time p.i. but not so much at early time likely results from lesserrequirements of smaller tumors (typically below 300 mm3, see FIG. 3 andFIG. 5) for neovascularization to provide the growth nutrients (for areview see 24).

[0135] Inhibition of LLC cells dissemination to the lungs was onlytransient as the survival rate from the AdmATF-treated group was onlyslightly extended (less than 30 days after tumor implantation) ascompared to that from the AdCO1-treated group (less than 25 days). Theeffects of AdmATF injection on tumor cells dissemination may beexplained either because the tumor cells were frozen following AdmATFinfection, and/or because few vessels were available for entry into thevasculature. That dissemination did eventually occur suggests that sometumor cells may have had already reached the vasculature at the time ofAdmATF injection. Alternatively, infection with E1-deleted adenovirusesis also typically associated with a rapid clearance of the infectedcells in C57BL/6 mice immunotolerant for the transgene product (29), andATF is a small molecule that exhibits a very short half-life in vivo .

[0136] Preclinical data indicate that the uPA/uPAR complex is criticallyinvolved in controlling cell migration, including that of endothelialcells. For example, an ATF-IgG fusion protein with an extended in vivohalf-life has been shown to inhibit angiogenesis and tumor growth in abFGF-enriched Matrigel murine model (23). The present study providesevidence that the antitumoral effects of uPA/uPAR antagonists areessentially exerted by controlling intratumoral and peripheralangiogenesis: whereas the antitumoral effects of AdmATF-mediated genedelivery may have been multifactorial as both tumor and endothelialcells are potential targets in the syngeneic tumor model, this is notthe case in the MDA-MB-231/athymic murine model because mATF is a poorantagonist of uPA/uPAR complex formation at the surface of human cells,including MDA-MB-231 (32). A remarkable feature that emerged in theMDA-MB-231 model was the efficacy of AdmATF in preventing tumor growth(FIG. 5) and neovascularization within and at the vicinity of the tumor(FIG. 6). In contrast, tumors infected with the control adenovirus werestill capable of “attracting” adjacent vessels. The antitumoralproperties of AdmATF are further illustrated in this model by thereduced efficacy of tumor establishment following infection.

[0137] Malignant tumors are life-threatening because they invade andabrogate the function of vital organs at distant sites, emphasizing theimportance of targeting angiogenesis to fight cancer (33; see also 34).First, growth of primary tumors relies on neovascularization to providethe required nutrients. Second, metastases have also been reported toundergo apoptosis in the absence of neovascularization (35).Furthermore, growing capillaries within the tumor are “leaky”: theyexhibit a fragmented basal membrane (36), a prerequisite for efficientpenetration of the tumor cells into the vasculature (37). The overallresults of this study demonstrate that significant antitumoral effectscan be achieved following a single intratumoral administration of arecombinant adenovirus expressing a potent antagonist of uPA/uPARfunction at the cell surface, and that these effects mostly result froman inhibition of angiogenesis. Applying this approach to invasive solidtumors is certainly attractive for cancer gene therapy because of thepleiotropic clinical effects expected following inhibition of tumorangiogenesis.

EXAMPLE 2 Gene Therapy With Angiostatin Inhibits Tumors In Vivo

[0138] Example 2 demonstrates that expression of the amino terminalfragment of human plasminogen (angiostatin K3) inhibits tumor growth invivo by blocking endothelial cell proliferation associated with amitosis arrest. The antitumoral effects that follow the local deliveryof the N-terminal fragment of human plasminogen (angiostatin K3) havebeen studied in two xenograft murine models. Angiostatin delivery wasachieved by a defective adenovirus expressing a secretable angiostatinK3 molecule from the CMV promoter (AdK3). In in vitro studies, AdK3selectively inhibited endothelial cell proliferation, and disrupted theG2/M transition induced by M-phase-promoting factors. AdK3-infectedendothelial cells showed a marked mitosis arrest that correlated withthe downregulation of the M-phase phosphoproteins. A single intratumoralinjection of AdK3 into pre-established rat C6 glioma or human MDA-MB-231breast carcinoma grown in athymic mice was followed by a significantarrest of tumor growth, that was associated with a suppression ofneovascularization within and at vicinity of the tumors. AdK3 therapyalso induced a 10-fold increase in apoptotic tumor cells as compared tocontrol adenovirus. The data support the concept that targetedanti-angiogenesis, using adenovirus-mediated gene transfer, represents apromising strategy for delivering anti-angiogeneic factors as bolusinjections of anti-angiogenic proteins still present unsolvedpharmacological problems.

Methods

[0139] Construction of AdK3. AdK3 is an E1-defective recombinantadenovirus that expresses the N-terminal fragment of human plasminogen(up to residue 333) from the CMV promoter. Human Plg cDNA was obtainedfrom plasmid PG5NM119. A fragment encoding the 18 aa signal peptide ofPlg, followed by the first 326 residues of mature Plg was firstsubcloned between the BamHI and ScaI sites of plasmid pXL2675. Asynthetic oligodeoxynucleotide encoding residues 327 to 333 followed bya stop codon was then added, prior to inclusion between the CMV promoterand the SV40 late polyadenylation signal. This expression cassette wasthen inserted between the EcORV and BamHI sites of plasmid pCO5 togenerate plasmid pCO5-K3. AdK3 was constructed in 293 cells byhomologous recombination between pCO5-K3 and ClaI-restricted AdRSVβgalDNA [25]. Individual plaques were isolated onto 293-derived cellmonolayers, amplified onto fresh 293 cells and viral stocks wereprepared as described [25]. AdCO1 is a control virus that is identicalto AdK3 except that it does not carry any expression cassette.

[0140] Cell lines maintenance and infection. C6 glioma cells (ATCCCCL-107) and MDA-MB 231 cells (ATCC HTB 26) were cultured in DMEM with10% of fetal calf serum (FCS). Viral infection was performed with 5%FCS. Human Microcapillary Endothelial Cells (HMEC-1) [49] were culturedin MCDB 131 supplemented with 20% of FCS, 1 mM L-glutamine, 1 μg/ml ofhydrocortisone, 10 ng/ml of epithelium growth factor and infection wasperformed in the same medium but with 10% of FCS and 3 ng/ml ofrecombinant human b-FGF (R&D system). The multiplicity of infection(MOI) was chosen as to obtain between 80% to 100% infected cells asjudged by X-GAL staining following infection with virus AdRSVβGal.

[0141] Western blot analysis. Subconfluent cells were infected with AdK3or AdCO1 at an MOI of 300 plaque-forming units (PFU)/cell. Cell culturesupernatants were collected 48 to 96 hr post-infection (p.i.). For invivo immunological analysis of the K3 angiostatin molecule, the tumorswere collected at day 10 p.i., frozen in liquid nitrogen, powdered,extracted with lysis buffer (10 mM NEM, 1% triton X100, 1 mM PMSF, 0.1MNH OH) and centrifuged at 12000 rpm at 4° C. The samples with 300 μg ofprotein were run in a 10% SDS-polyacrylamide gel, prior to beingtransferred onto a nitrocellulose membrane (Schleicher & Schuell). 100ng human Plg (Stago) was run as a control. After 2 hr incubation inblocking buffer (TBS-5% milk-0.05% Tween 20), the membranes wereincubated for 1 hr with anti-human Plg MAb A1D12 [50], 1 hr with ahorseradish peroxidase-conjugated goat anti-mouse serum (Biosys). Afterwashing, the membranes were detected with ECL bioluminescence kit(Amersham, UK). To detect the MPM-2 phosphoepitope, the extracts wereprepared from the HMEC-1 cell 96 hr p.i. and probed with the specificmitotic MPM-2 MAb (DAKO).

[0142] Proliferation assay. Tumor or HMEC-1 cells were infected withAdK3 or AdCO1 at the indicated MOI for 12 hr. The cells were collectedwith 1 mM EDTA, washed twice with PBS and resuspended. They were seededinto 96-well culture plates (5000 cells/well) and cultured for 72 hr. Inaddition, HMEC-1 cells were cultured in MCDB131 medium containing 40, 20or 10% supernatant of AdK3 or AdCO1-transduced C6 glioma cells.Supernatants from virally-infected C6 cells were collected 96 hr p.i.,heated 30 min at 56° C. in order to inactivate the virus, concentrated10 times and dialyzed against PBS. Cells were quantified with a cellproliferation assay kit using a MTS tetrazolium compound (Promega).

[0143] Formation of capillary tube in a fibrin matrix model. This modelwas devised according to the method of Pepper et al [51] using CalfPulmonary Artery Endothelial cells (CPAE) (ATCC CCL 209) infected for 12hr with AdK3 or AdCO1 at an MOI of 600.

[0144] Whole blood lysis assay. Whole blood clot lysis was performed bymixing 80 U/ml of tissue-plasminogen activator, 250 μl of culturesupernatant obtained 4 days p.i. with AdK3 or AdCO1, and 500 μl ofcitrate-anti-coagulated whole blood collected from healthy donors.Coagulation was triggered by adding 1 U/ml of thrombin and of 12 mMCa++. The extent of clot lysis was determined by lysis time and byfollowing the kinetics of soluble D-Dimers as described [52].

[0145] Immunoflow cytometry. HMEC-1 were infected for 96 hr with AdK3 orAdCO1 at an MOI of 300 PFU/cell. The cells were collected, permeabilizedwith triton X100, incubated with iodide propidium (20 μg/ml) andribonuclease A (100 μg/ml) for 30 min at room temperature to label DNA,prior to incubation with mitotic MPM-2 antibody as described [53].FITC-conjugated anti-mouse IgG antibodies were used to detect MPM-2phosphoepitope. The experiment was performed in a Coulter EPICS ProfileII flow cytometer and the data were analysed by Multicycle software(Phoenix Flow Systems, San Diego, Calif.).

[0146] Athymic murine models. Cultured C6 glioma cells and MDA-MB-231cells were harvested, washed, resuspended in PBS at 1.5×1 07 and0.25×10⁷ cells/ml respectively and a volume of 200 μl was subcutaneouslyinjected into the dorsa of 6-7 weeks old nude mice. When the tumors hadreached a volume of 20 mm³, the animals received an intratumoralinjection of 10⁹ PFU of either AdK3 (n=6), or AdCOI (n=6), or PBS (n=6).Tumor size was monitored until day 10 p.i. for the C6 glioma model, andday 42 p.i. for the MDA-MB-231 model.

[0147] To assess the effect of AdK3 infection on tumor establishment andprogression, MDA-MB-231 and C6 cells were infected for 24 hr at an MOIof 50 and 100 PFU/cell, respectively, prior to subcutaneous inoculationinto the dorsa of nude mice (n=6). Infected MDA-MB-231 cells are lesstumorigenic than infected C6 cells so 80 μl ice-cold Matrigel (BectonDickinson) had to be added to 120 μl of PBS prior to subcutaneousimplantation (10⁶ MDA-MB-231 or 0.25×10⁶ C6 cells). Tumor establishmentand growth were followed until day 25 (MDA-MB-213) or day 22 (C6) p.i. AStudent's t-test was used for statistical analysis.

[0148] Immunohistochemistry. Tumor tissues were fixed in alcoholformalin acetic acid, embedded in paraffin and 5 pm sections wereprepared. After toluene treatment and rehydration, the sections werepretreated three times for 5 min in a microwave oven in 10 mM citratebuffer (pH 6.0), quenched by 3% H₂O₂ for 5 min to remove endogenousperoxidase activity, washed in PBS, then incubated with a rabbitpolyclonal serum raised against human von Willebrand factor (vWF; Dako,dilution 1:200) for 60 min. After 3 washes, the sections were incubatedwith biotinylated goat anti-rabbit IgG antibodies for 30 min., washed,and incubated with streptavidin-peroxidase for 30 min. prior to additionof 3-Amino-9-ethyl-carbazole. Meyer's hematoxylin was used forcounterstaining. Apoptotic cells within the section were detected by akit using a terminal deoxynucleotidyl transferase-mediated dUTP-biotinnick end labeling method (TUNEL) (Boehringer Mannheim). Forproliferating cell nuclear antigen (PCNA) staining procedure included abiotinylated mouse anti-PCNA antibody (Pharmingen, dilution 1:100)followed by streptavidin peroxidase and substrate revelation.

Results

[0149] Molecular characterization of AdK3. Recombinant AdK3 carries aCMV-driven N-terminal fragment of human Pig that includes the firstthree kringle domains of the angiostatin molecule [47], whereas AdCO1 isan “isogenic” control adenovirus that does not encode any expressioncassette (FIG. 7A). Secretion of the K3 molecule in the culture media2-3 days after infection with AdK3 was demonstrated for HMEC-1, C6 andMDA-MB-231 cells by Mab A1D12 immunoblotting, whereas no signal wasdetected following infection with AdCO1 (FIG. 7B). The secretedimmunoreactive peptide appeared as a doublet with a molecular weight of36 and 38 kDa, most likely reflecting a different extent ofN-glycosylation at Asn²⁸⁹ as described for Plg [54, 55].

[0150] Functional characterization of AdK3. Transduction of HMEC-1 byAdK3 resulted in an inhibition of bFGF-stimulated proliferation in adose-dependent manner at day 3 p.i.: 30% at an MOI of 50, 74% at an MOIof 150, and 97% at an MOI of 300, in sharp contrast to the cellsinfected with AdCO1 (P<0.005). AdK3 did not affect MDA-MB-231 and C6cell proliferation (FIG. 8A). To assess the paracrine potential of theK3 molecule to exert these effects, virus-free culture media fromvirally-infected C6 glioma cells were added to HMEC-1 cells. Asillustrated in FIG. 8A, we did observe a dose-dependent inhibition ofHMEC-1 cell proliferation by C6 cell-secreted angiostatin (p<0.001). Theaddition of AdK3 also significantly inhibited the capillary formation ofCPAE cells in fibrin gel with a 55% mean reduction (not shown).Moreover, whole blood clot lysis induced by tPA was not inhibited by theaddition of cell culture supernatants from AdK3-infected C6 cells, andthe generation of D-Dimers was basically unchanged during the firstthree hours (1200 ng/ml versus 1150 ng/ml).

[0151] AdK3 inhibits mitosis of endothelial cells. To determine ifangiostatin is able to block the mitosis of HMEC-1, a flowimmunocytometry analysis was performed with the cells labeled with MAbMPM-2 that binds to the phosphorylated proteins specifically presentduring the M-phase, together with concurrent DNA staining. The resultsshowed that mitosis of AdK3-infected HMEC-1 cells was decreased by 82%relative to AdCO 1 infection: only 5% of HMEC-1 cells within the G2/Mpic scored positive for MPM-2 following infection with AdK3 as comparedto 27% following AdCO1 control infection (FIG. 8C). Western blotanalysis was performed from HMEC-1 extracts in order to detect MPM-2positive proteins as at least 16 mitotic phosphoproteins were usuallyrevealed by MPM-2 with an apparent molecular weight ranging from 40 tomore than 200 kDa. As compared to control extracts from non-infected orAdCO1-infected HMEC-1 cells, extracts from AdK3-infected cells exhibiteda markedly reduced level of MPM2-reactive phosphoproteins (FIG. 8B).

[0152] AdK3 inhibits tumor growth. To induce local secretion ofangiostatin, a single dose of 109 PFU of AdK3 was injected into 20 mm³pre-established human MDA-MB-231 breast carcinoma and rat C6 gliomatumors grown in athymic mice, and tumor growth was monitored. As shownin FIG. 9A, C6 tumors from the AdK3-injected group were significantlysmaller than those from the AdCO1 or the PBS control groups: at day 10p.i., AdK3-injected tumors had reached a mean volume of 278±14 mm³versus 1403±142 mm³ or 1583±259 mm³ for AdCO1- and PBS-injected tumors,respectively (p<0.05). This 80% inhibition correlated with the detectionof angiostatin-immunoreactive material (FIG. 7C). As shown in FIG. 9B,tumor growth was similarly inhibited (85%) in the MDA-MB-231 tumor modelat day 42 p.i.: 80±4 mm³ for AdK3-treated tumors versus 563±137 mm³ forAdCO1- and 530±69 mm³ for PBS-injected tumors respectively (p<0.05).

[0153] AdK3 inhibits angiogenesis and induces tumor cell apoptosis invivo. C6 tumors infected with AdCO1 appeared much more vascularized thantheir AdK3-infected counterparts (FIG. 10, panels A-B). Intratumoralangiogenesis was thus assessed by vWF-immunostaining of tumor sectionsas described [28]. vWF-positive hotspots were first localized at lowmagnification, and vWF-positive vessels were then counted at 200×magnification (FIG. 10, panels E-F). The results indicated a markedreduction of intratumoral vascularization within AdK3-injected tumors(5±2 vWF-positive vessels per field) as compared to the AdCO1-injectedcontrol (14±4; n=5, p<0.005). Tumors in the PBS-injected group exhibitedan identical number of vessels (14±3) indicating that the infectionconditions used did not interfere with tumor angiogenesis. At themacroscopic level, C6 tumors injected with AdK3 displayed littleperipheral neovascularization as compared to their AdCO1-treatedcounterparts (FIG. 10, panels C-D). Similar results were obtained withinMDA-MB-231 tumor sections (4.8±1.2 vWF-immunoreactive vessels/field forAdK3 versus 15.6±3 for AdCO1, p=0.02).

[0154] Tumor cell apoptosis was then quantified in situ with the C6tumor samples by the TUNEL method (see Methods). The results indicated amarked increase of apoptotic cells in the AdK3-injected C6 tumors 10days p.i. (20±9 versus 1-2 apoptotic cells per field for control tumors,p<0.001) (FIG. 10, panels G-H). In contrast, the tumor cellproliferation rate was not different among the three animal groups asassessed by PCNA immunostaining (not shown). Ad-angiostatin therapyinduced a 10 fold increase in apoptotic tumor cells without affectingthe proliferation of these cells, similar to the reported resultsobtained by daily injections of purified angiostatin.

[0155] AdK3 inhibits tumorigenesis. To determine whether inhibition oftumor angiogenesis attenuated tumorigenesis, MDA-MB-231 and C6 cellswere first infected for 24 hr prior to injection into the dorsa of nudemice. After 5 days, all the mice from the AdCO1-infected group developedhypervascularized C6 tumors with an average size of 27.4 +3.41 mm³,whereas 20% of animals from the AdK3-infected group remained tumor freeafter 12 days (FIG. 11). The remaining animals exhibited very smalltumors (average size of 0.42±0.05 mm³) that were hardly vascularized.After 22 days, the tumors that were observed within the AdK3 group wereat least 5-fold smaller than those from the AdCO1 group (n=5, p<0.005;FIG. 11). Similar observations were made with the MDA-MB-231 tumor model(not shown).

Discussion

[0156] Angiostatin has been shown to be a physiopathological inhibitorof angiogenesis secreted by primary tumors, driving the metastasis intoa dormant state. It was therefore tempting to assess the therapeuticpotential of angiostatin on primary tumors. However, systemic andintraperitoneal bolus injections of human angiostatin have underlineddifficult pharmacological problems because angiostatin is rapidlycleared from the circulation [46]. A prolonged exposure of purifiedangiostatin at high doses was indeed required to maintain cytostaticintratumoral concentrations of angiostatin [46]. It was not clear thatdirect transduction of the tumor and the surrounding tissue with arecombinant virus encoding an angiostatin cDNA would represent a moreefficient method of achieving constant intratumoral concentrations ofangiostatin. Adenoviruses are appropriate vectors in such a strategy asthey can efficiently express their transgene at therapeutic levels inboth proliferating and non-proliferating cells (for a review see [37]),allowing to target a wide area for angiostatin production. Thus, adefective adenovirus that expresses the N-terminal fragment (aa 1-333)from human Plg, including its pre-activation peptide and kringles 1 to 3(AdK3) was constructed.

[0157] The use of Mab A1D12, which is specific to human Plg [50] firstdemonstrated an efficient secretion of angiostatin in the culture mediaof cells infected with AdK3. The inclusion of the N-terminalpre-activation peptide in the angiostatin molecule did not affect itsanti-angiogenic activity since AdK3- but not AdCO1-infected endothelialcells showed a marked, dose-dependent, arrest in proliferation in vitro(FIG. 8A). Furthermore, the proliferation of MDA-MB-231 or C6 tumorcells was not affected by AdK3-infection demonstrating the restrictedaction of angiostatin for endothelial cells. Virus-free supernatantsfrom AdK3-infected tumor cell culture also inhibited endothelial cellproliferation, illustrating the paracrine effect of angiostatin secretedby transduced-tumor cells.

[0158] Because the kringle domains are important for Plg binding tofibrin and fibrin degradation, it was essential to analyze the effect ofthis therapy in thrombolysis, a physiological protection againstthrombosis in vivo. The angiostatin secreted in the culture mediumfailed to inhibit tPA-induced whole blood clot lysis in vitro. Althoughthis experiment has not excluded the deleterious competition betweenangiostatin and Plg to bind to fibrin during thrombolysis in vivo, itindicates that an angiostatic effect could be achieved at aconcentration far below that required for abrogatingplasminogen-dependent thrombolysis in vivo. This may also suggest thatendothelial cells exhibit a receptor that recognizes angiostatin and notintact Pig.

[0159] Flow cytometry analysis of endothelial cells infected with AdK3demonstrated a complete disappearance of the mitotic population positivefor MPM-2 MAb [56]. Immunoblot analysis revealed that M-phasephosphoproteins reactive to MPM-2 MAb were indeed downregulated inarigiostatin-treated endothelial cells, in sharp contrast with controlendothelial cells. This observation should be helpful to define themechanism by which angiostatin abrogates the proliferation ofendothelial cells. We also showed that angiostatin disrupted the G2/Mtransition induced by M-phase-promoting factor (MPF), composed of cdc2and its associated regulatory subunit, cyclin B [57]. MPF phosphorylatedproteins, reactive with MPM-2 MAb, are involved in major alterations ofcellular structures and activities for an efficient progression tomitosis. The reason why active MPF was lacking in AdK3-transducedendothelial cells must be further investigated.

[0160] A single intratumoral injection of AdK3, but not of AdCO1 wasshown to dramatically inhibit primary tumor growth in twopre-established xenograft murine models. This inhibitory effect on tumorgrowth was tightly correlated with a markedly decreased vascularizationwithin, and at the vicinity of the tumors (FIG. 10), together with thedetection of angiostatin-immunoreactive material in the tumor extracts(FIG. 7C). C6 glioma is a highly vascularized tumor due to its VEGFoverexpression [58]. Interestingly, the AdK3-transduced C6 gliomaapparently failed to establish a vascular network within the tumor massto support rapid and extensive growth (FIG. 10), and this failuretranslated to more than 80% inhibition of tumor growth. vWFimmunostaining of tumor sections also revealed a significant reductionof neoangiogenesis in the AdK3-treated tumors: well formed vessels witha mature lumen were frequently observed in control C6 tumors, but not inAdK3-treated C6 glioma (FIG. 10). This decrease in vessel density wasassociated with a 10-fold increase in tumor cells apoptosis and noapparent modification of the tumor cell proliferation index, probablybecause (i) of the lack of endothelial-derived paracrine factors, (ii) areduction in nutrient support, and (iii) hypoxia triggered p53-dependentapoptosis of the tumor cells [59, 60]. In the MDA-MB-231 breastcarcinoma model, a single intratumoral injection of AdK3 similarlyinduced a remarkable inhibition of tumor angiogenesis and growth.

[0161] In the course of this study, AdK3-transduced C6 and MDA-MB-231cells exhibited a lower tumorigenic potential as reflected by aprolonged delay for AdK3-infected cells to develop into visible tumorsfollowing implantation.

[0162] Angiostatic therapy using recombinant adenoviruses has been shownto be experimentally plausible and efficient. The possibility ofdelivering more than one angiostatic factor could also synergize toarrest tumor growth. It is also envisioned that its association withcytotoxic approaches may be particularly potent to improve the clinicaloutcome of malignant diseases.

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[0224] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

[0225] It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription.

[0226] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

What is claimed is:
 1. A method for inhibiting growth of a tumor comprising introducing into the tumor a defective adenovirus vector comprising a gene encoding an anti-angiogenic factor operably associated with an expression control sequence that provides for expression of the anti-angiogenic factor in a cell of the tumor.
 2. The method according to claim 1, wherein the tumor is a lung carcinoma or a breast carcinoma.
 3. The method according to claim 1, wherein the anti-angiogenic factor comprises a sequence of an amino terminal fragment of urokinase having an EGF-like domain, with the proviso that the factor is not urokinase.
 4. The method according to claim 3, wherein the anti-angiogenic factor is an amino terminal fragment of urokinase having an amino acid sequence of urokinase from about amino acid residue 1 to about residue
 135. 5. The method according to claim 4, wherein the urokinase is murine urokinase.
 6. The method according to claim 4, wherein the urokinase is human urokinase.
 7. The method according to claim 1, wherein the anti-angiogenic factor is angiostatin.
 8. The method according to claim 7, wherein the angiostatin comprises kringles 1 to
 3. 9. The method according to claim 7, wherein the angiostatin is an amino terminal fragment of plasminogen (Plg) having an amino acid sequence of plasminogen from about amino acid residue 1 to about residue
 333. 10. The method according to claim 9, wherein the plasminogen is human plasminogen.
 11. A method for inhibiting growth or metastasis, or both, of a tumor comprising introducing a vector comprising a gene encoding an amino terminal fragment of urokinase having an EGF-like domain into the tumor, with the proviso that the gene does not encode urokinase, wherein the gene is operably associated with an expression control sequence that provides for expression of the gene in a cell of the tumor.
 12. The method according to claim 11, wherein the amino terminal fragment of urokinase has an amino acid sequence of urokinase from about amino acid residue 1 to about residue
 135. 13. The method according to claim 12, wherein the urokinase in murine urokinase.
 14. The method according to claim 12, wherein the urokinase in human urokinase.
 15. A defective adenovirus vector comprising a gene encoding an anti-angiogenic factor operably associated with an expression control sequence.
 16. The virus vector according to claim 15, wherein the anti-angiogenic factor comprises a nucleic acid sequence of an amino terminal fragment of urokinase having an EGF-like domain, with the proviso that the factor is not urokinase.
 17. A defective adenovirus vector comprising a gene encoding an amino terminal fragment of urokinase having an EGF-like domain, with the proviso that the gene does not encode urokinase.
 18. The virus vector according to claim 17, wherein the amino terminal fragment of urokinase has an amino acid sequence of urokinase from amino acid residue 1 to about residue
 135. 19. The virus vector according to claim 18, wherein the urokinase is murine urokinase.
 20. The virus vector according to claim 18, wherein the urokinase is human urokinase.
 21. The virus vector according to claim 15, wherein the anti-angiogenic factor is angiostatin.
 22. The virus vector according to claim 21, wherein the angiostatin comprises kringles 1 to
 3. 23. The virus vector according to claim 21, wherein the angiostatin comprises a nucleic acid sequence of an amino terminal fragment of plasminogen having an amino acid sequence of plasminogen from amino acid residue 1 to about residue
 333. 24. The virus vector according to claim 23, wherein the plasminogen is human plasminogen.
 25. A pharmaceutical composition comprising a virus vector of any one of claims 15-24 and a pharmaceutically acceptable carrier.
 26. Use of the virus vector of any one of claims 15-24 in the manufacture of a medicament for inhibiting growth of a tumor.
 27. Use of the virus vector of any one of claims 16-20 in the manufacture of a medicament for inhibiting growth, or metastasis, or both of a tumor.
 28. Use of the virus vector of any one of claims 21-24 in the manufacture of a medicament for inhibiting tumor growth and inducing apoptosis.
 29. Use of a vector comprising a gene encoding an amino-terminal fragment of urokinase having an EGF-like domain, with the proviso that the gene does not encode urokinase, operably associated with an expression control sequence that provides for expression of the anti-angiogenic factor in the manufacture of a medicament for inhibiting growth or metastasis, or both, of a tumor.
 30. The use according to any of claims 26-29, wherein the tumor is a lung carcinoma or a breast carcinoma.
 33. The method according to claim 31, wherein the anti-angiogenic factor comprises an amino terminal fragment of urokinase comprising an EGF-like domain, with the exception that the anti-angiogenic factor is not urokinase.
 34. The method according to claim 33, wherein the anti-angiogenic factor is an amino terminal fragment of urokinase comprising an amino acid sequence of urokinase from about amino acid residue 1 to about residue
 135. 35. The method according to claim 34, wherein the urokinase is murine urokinase.
 36. The method according to claim 34, wherein the urokinase is human urokinase.
 37. The method according to claim 31, wherein the anti-angiogenic factor is angiostatin.
 38. The method according to claim 37, wherein the angiostatin comprises kringles 1 to
 3. 39. The method according to claim 37, wherein the angiostatin is an amino terminal fragment of plasminogen (Plg) comprising an amino acid sequence of plasminogen from about amino acid residue 1 to about residue
 333. 40. The method according to claim 39, wherein the plasminogen is human plasminogen.
 41. A method for inhibiting growth and/or metastasis of a tumor comprising introducing a vector comprising a gene encoding an amino terminal fragment of urokinase comprising an EGF-like domain into the tumor, with the exception that the gene does not encode urokinase, wherein the gene is operably associated with an expression control sequence that provides for expression of the gene in a cell of the tumor.
 42. The method according to claim 41, wherein the amino terminal fragment of urokinase comprises an amino acid sequence of urokinase from about amino acid residue 1 to about residue
 135. 43. The method according to claim 42, wherein the urokinase is murine urokinase.
 44. The method according to claim 42, wherein the urokinase is human urokinase.
 45. A defective adenovirus vector comprising a gene encoding an anti-angiogenic factor operably associated with an expression control sequence.
 46. The defective adenovirus vector according to claim 45, wherein the anti-angiogenic factor comprises an amino terminal fragment of urokinase comprising an EGF-like domain, with the exception that the anti-angiogenic factor is not urokinase.
 47. A defective adenovirus vector comprising a gene encoding an amino terminal fragment of urokinase comprising an EGF-like domain, with the exception that the gene does not encode urokinase.
 48. The defective adenovirus vector according to claim 47, wherein the amino terminal fragment of urokinase comprises an amino acid sequence of urokinase from about amino acid residue 1 to about residue
 135. 49. The defective adenovirus vector according to claim 48, wherein the urokinase is murine urokinase.
 50. The defective adenovirus vector according to claim 48, wherein the urokinase is human urokinase.
 51. The defective adenovirus vector according to claim 45, wherein the anti-angiogenic factor is angiostatin.
 52. The defective adenovirus vector according to claim 51, wherein the angiostatin comprises kringles 1 to
 3. 53. The defective adenovirus vector according to claim 51, wherein the angiostatin comprises an amino terminal fragment of plasminogen comprising an amino acid sequence of plasminogen from about amino acid residue 1 to about residue
 333. 54. The defective adenovirus vector according to claim 53, wherein the plasminogen is human plasminogen.
 55. A pharmaceutical composition comprising the defective adenovirus vector according to claim 45 and a pharmaceutically acceptable carrier.
 56. A pharmaceutical composition comprising the defective adenovirus vector according to claim 47 and a pharmaceutically acceptable carrier. 